Synthesis of nano-sized β-tricalcium phosphate via wet precipitation

Nano-size β-tricalcium phosphate powders with average grain size of 50 nm were prepared by the wet chemical precipitation method with calcium nitrate and diammonium hydrogen phosphate as calcium and phosphorus precursors, respectively. The pH of the system was maintained at 8 and 10.8 by adding of sodium hydroxide. Filtered cake was dried at 80°C and calcined at 700°C. The dried and calcined powders were characterized using X-ray diffractrometry (XRD), Fourier transform infrared spectroscopy (FTIR), inductively coupled plasma atomic emission spectroscopy (ICPAES) and scanning electron microscopy (SEM).


I. Introduction
β-tricalcium phosphate [β-Ca 3 (PO 4 ) 2 ] (β-TCP) and hydroxyapatite [Ca 10 (PO 4 ) 6 (OH) 2 ] (HA) powders are widely applied in the biomedical fields because of their biocompatibility and osteoconductivity [1,2].HA is thermodynamically the most stable phase in physiological conditions and has the ability for direct chemical bonding to the bone, while β-TCP is found to be resorbable in vivo with new bone growth replacing the implanted β-TCP [3,4].Tricalcium phosphate is one of the most important biomaterials based on phosphates, currently recognized as ceramic material that significantly simulates the mineralogical structure of bone [5][6][7][8].
TCP has three polymorphic forms: β, α and α′.The latter is of no interest because it transforms into the α-form during cooling.β-TCP is stable at room temperature and reconstructively transforms at 1125°C into α-TCP, which is metastably retained until room temperature during the cooling [9,10].
β-TCP is the low-temperature phase in the CaO-P 2 O 5 phase diagram.As it was mentioned, β-TCP transforms to α-TCP at 1125°C and above this, up to 1430°C, α-TCP is a stable phase.Above 1430°C, the super α-TCP form becomes stable until the melting point of 1756°C.
The ideal Ca/P ratio of β-TCP is 1.5 and the theoretical density is 3.17 g/cm 3 as reported by Guelcher and Hollinger [11].
Often tribasic calcium phosphate is mistaken for β-tricalcium phosphate.According to Metsger et al. [12], tribasic calcium phosphate is a nonstoichiometric compound often bearing the formula of hydroxyapatite [Ca 10 (PO 4 ) 6 (OH) 2 ].TCP is a resorbable temporary bone space filler material.When implanted, TCP will interact with body fluids and form HA in accordance with the following equation: The reaction rate will decrease with increasing pH of the local solution and further increase the solubility of TCP.Theoretically, resorbable TCP is an ideal implant material.After implantation, TCP will degrade with time and be replaced with natural tissues.It leads to the regeneration of tissues instead of their replacement and so solves the problem of interfacial stability [11,12].β-TCP powders are reportedly prepared by liquid-solution methods, such as sol-gel, hydrothermal, micro emulsion and precipitation, as well as gas phase reactions [13][14][15].
Synthesis of calcium phosphate nanopowder is relevant in biological fields, because the dimensions of large biomolecules such as proteins and DNA as well as those of many important subcellular structures fall in the size range between 1 and 1000 nm [16,17].In the present research work, a wet chemical reaction such as the precipitation method was carried out to prepare β-TCP.

Powder synthesis
Detail procedure and experimental conditions for preparation of β-TCP powder by wet chemical process is shown in Fig. 1. β-TCP nanopowders were synthesized by the reaction of calcium nitrate tetra-hydrate (Ca(NO 3 ) 2 ×4H 2 O, 98%, Merck) with diammonium hydrogen phosphate ((NH 4 ) 2 HPO 4 , 99%, Merck).Briefly, 500 mL of 0.4 mol (NH 4 ) 2 HPO 4 solution with pH = 4 was vigorously stirred at room temperature, and 500 mL of 0.6 mol Ca(NO 3 ) 2 with pH = 7.3 was added drop wise over 150-200 min to produce a white precipitate.Throughout the mixing process the pH of the system was maintained at pH = 8 (sample A) and pH = 10.8 (sample B) by adding of 0.1 M sodium hydroxide (NaOH, 99%, Merck).The obtained white suspension was then stirred for 12 h.The synthesized precipitate was washing with distilled water and then with 100% ethanol to improve the dispersion characteristics.The suspension was filtered in a filter glass with application of mild suction.After filtration the compact, sticky filter cake, was dried at 80°C for 24 h.The as-dried powders were crushed by using mortar and pestle and calcined in alumina crucible at 700°C for 2 h.

Characterization
Evaluation of crystalline phases was investigated by X-ray diffractometer (Siemens, model D-500) using CuKα radiation.Silicon powder was used as the standard material for semi-quantitative analysis of precipitated phases.The mean crystallite size (D) was calculated from the XRD line broadening measurement from the Scherrer equation [18]: where λ is the wavelength of the used Cu Kα radiation, β is the full width at the half maximum of the β-TCP line and θ is the diffraction angle.
Infrared spectra were performed by FTIR Bomem (model MB100, Quebec, Canada) in 400-4000 cm -1 wavenumber region.For infrared spectroscopy, samples were pulverized and mixed with a given amount of potassium bromide (KBr) and pressed in very thin tablets.
The Ca/P ratio of the dried powder was measured by inductively coupled plasma (ICP) atomic emission spectroscopy (model Varian).No differences were observed in the Ca/P values between the as-dried and calcined powders.

Powder synthesis
Detail procedure and experimental conditions for preparation of -TCP powder by wet chemical process is shown in Fig. 1. -TCP nanopowders were synthesized by the reaction of calcium nitrate tetrahydrate (Ca(NO 3 ) 2 ×4H 2 O, 98%, Merck) with diammonium hydrogen phosphate ((NH 4 ) 2 HPO 4 , 99%, Merck).Briefly, 500 mL of 0.4 mol (NH 4 ) 2 HPO 4 solution with pH = 4 was vigorously stirred at room temperature, and 500 mL of 0.6 mol Ca(NO 3 ) 2 with pH = 7.3 was added drop wise over 150-200 min to produce a white precipitate.Throughout the mixing process the pH of the system was maintained at pH = 8 (sample A) and pH = 10.8 (sample B) by adding of 0.1 M sodium hydroxide (NaOH, 99%, Merck).The obtained white suspension was then stirred for 12 h.The synthesized precipitate was washing with distilled water and then with 100% ethanol to improve the dispersion characteristics.The suspension was filtered in a filter glass with application of mild suction.After filtration the compact, sticky filter cake, was dried at 80°C for 24 h.The as-dried powders were crushed by using mortar and pestle and calcined in alumina crucible at 700°C for 2 h.

Characterization
Evaluation of crystalline phases was investigated by X-ray diffractometer (Siemens, model D-500) using CuK radiation.Silicon powder was used as the standard material for semi-quantitative analysis of precipitated phases.The mean crystallite size (D) was calculated from the XRD line broadening measurement from the Scherrer equation [18]: where is the wavelength of the used Cu K radiation, is the full width at the half maximum of the -TCP line and is the diffraction angle.
Infrared spectra were performed by FTIR Bomem (model MB100, Quebec, Canada) in 400-4000 cm -1 wavenumber region.For infrared spectroscopy, samples were pulverized and mixed with a given amount of potassium bromide (KBr) and pressed in very thin tablets.
The Ca/P ratio of the dried powder was measured by inductively coupled plasma (ICP) atomic emission spectroscopy (model Varian).No differences were observed in the Ca/P values between the as-dried and calcined powders.

III. Results and discussion
The reactions involved in the formation of β-TCP during the chemical precipitation can be expressed as follows: The effect of the solution pH on the composition of the precipitates was investigated by ICP method, and analysis was performed on the precipitates dried at 100°C for 24 h.It can be seen, Table 1, that the Ca/P value was 1.51 and 1.59 when the pH of the precursor solution was 8.0 and 10.8, respectively.The unreacted precursors are believed to be washed out during filtering process.
Figure 2 shows SEM micrograph of the samples A and B after filtering and before calcination.It can be seen that the as-prepared powders consist of aggregated particles with broad size distribution.

FTIR spectroscopy
The FTIR spectrum of the powder prepared at pH = 8 is shown in Fig. 3.The characteristic absorption bands at 3433 and 1631 cm -1 are attributed to adsorbed water.The bands at 900-1200 cm -1 were the stretching mode of PO 4 3-group.The sharp peaks at 561 and 607 cm -1 represent the vibration peaks of PO 4 3-in β-TCP [19,20].The FTIR spectrum of the sample B, prepared at pH = 10.8, is shown in Fig. 4. The broad bands at 3433 cm -1 and 1642 cm -1 were attributed to adsorbed water, while sharp peak at 3572 cm -1 appeared due to the stretching vibration of the lattice OH -ions.The peak at 633 cm -1 was assigned to the O-H deformation mode.The characteristic bands due to vibration of PO 4 3-group appeared at 470, 570, 964 and 1046 cm -1 .The observation of the asymmetric P-O stretching vibration of the PO 4 3-bands at 964 cm -1 as a distinguishable peak, together with the sharp peaks at 633, 602 and 570 cm -1 correspond to the triply degenerate bending vibrations of PO 4 3 in hydroxyapatite.Our FTIR results were similar to the literature data [21][22][23].

XRD analysis
The XRD analysis was performed using the X-ray diffractometer.The straight base line and sharp peaks of the diffractogram in Fig. 5 confirmed that the products were well crystallized.The XRD pattern in Fig. 5 indicated that the sample A contains mostly β-TCP.On the other side, both crystalline phase (tricalcium phosphate and hydroxyapatite) exist in the sample B. XRD pattern also reveals that the as-dried powder B has a poorly crystallized hydroxyapatite phase.The average crystallite size, determined by Scherrer equation, of the sample A and B is 50 and 80 nm, respectively.
It is important to note that even there are many phases in addition to the hydroxyapatite and β-tricalcium phosphate, which have Ca/P ratios of 5/3 and 3/2, respectively (such as mono-, di-, tri-, and tetra-calcium phosphates), they were not detected in the investigated powders.Park and Lakes [24] reported that the stability in solution generally increases with increasing Ca/P ratios and that different phases can be used in different applications depending upon whether a degradable or a bioactive material is desired.Thus, our results confirmed that with increasing pH up to 10.8 it is possible to increase the Ca/P ratio to 1.59 and change phase composition of the synthesized powders.

SEM analysis
The morphologies of the TCP powder precipitated at pH = 8 was shown in Fig. 6 at two different magnifications.The TCP powder is highly agglomerated with almost spherical particles having average size of 150 nm.The necking among the particles was apparent due to localized sintering at 700°C. Figure 7 shows aggregates of sample B (synthesized at pH = 10.8) at two different magnifications.The powder is also highly agglomerated, but it consists of smaller particles, 80-150 nm.

V. Conclusions
In this study, the synthesis of biocompatible nanosized β-TCP powder via wet precipitation method and using calcium and phosphorous precursors is reported.It is shown that high purity products, β-TCP nanopowder and hydroxyapatite/β-TCP composite, could be obtained by this simple process.The sample prepared at pH = 8 is β-TCP powder with Ca/P ratio of 1.51, whereas the powder prepared at pH = 10.8 has hydroxyapatite/ β-TCP composite structure and Ca/P ratio 1.59.Thus, our results confirmed that with increasing pH up to 10.8 it is possible to increase the Ca/P ratio to 1.59 and change phase composition of the synthesized powders.The prepared powders could be widely used for biomedical applications.

Figure. 1 .
Figure. 1. Flowchart of wet-chemical precipitation process used for synthesis of β-TCP bioceramic powders

Figure. 1 .
Figure. 1. Flowcharts of wet-chemical precipitation process used for synthesis of -TCP bioceramic powders