V. Rodríguez-Lugo*a,
E. Salinas-Rodrígueza,
R. A. Vázqueza,
K. Alemánb and
A. L. Rivera*c
aUniversidad Autónoma del Estado de Hidalgo, Área Académica de Ciencias de la Tierra y Materiales, Carretera Pachuca-Tulancingo Km. 4.5, C.P. 42184, Pachuca, Hidalgo, Mexico. E-mail: ventura.rl65@gmail.com
bUniversidad Autónoma del Estado de Hidalgo, Área Académica de Computación y Electrónica, Carretera Pachuca-Tulancingo Km. 4.5, C.P. 42184, Pachuca, Hidalgo, Mexico
cInstituto de Ciencias Nucleares, Centro de Ciencias de la Complejidad, Universidad Nacional Autónoma de México, Apartado Postal 70-543, Deleg. Coyoacán, Mexico City, C.P. 04510, Mexico. E-mail: anarivera2000@gmail.com
First published on 23rd January 2017
Hydroxyapatite HAp, Ca10(PO4)6(OH)2, was successfully synthesized using a hydrothermal method using β-tricalcium phosphate (β-TCP) and CaO from the starfish Mellita eduardobarrosoi sp. nov. The goal of this research was to synthesize a material with better characteristics, such as a high proportion of the HAp phase, homogeneous dimensions, and a good Ca/P ratio. To get it, different temperatures, concentration ratios and reaction times were tested. Synthesized HAp materials were characterized by Scanning Electron Microscopy (SEM), X-ray powder diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). XRD showed that the proportion of the HAp phase tends to increase as the temperature and reaction times grow, which is corroborated by SEM characterization, where homogeneous in size HAp fibers were identified. Absorption bands related to the functional groups PO4 and OH, as well as the characteristics of HAp, were found by FTIR. From the results obtained, optimal HAp is achieved at a temperature of 250 °C and a reaction time of 48 hours, producing a greater proportion of fibers with very homogeneous lengths and thicknesses, a Ca/P ratio of 1.65 (near stoichiometric), a higher crystalline degree, and a ratio of 3:1 of β-TCP and CaO.
The term “apatite” is applied to a large group of mineral compounds featuring a general formula:9 A10(BP4)6X2, where A can be Ca, Sr, Ba, Cd, or other rare earth elements; BP4 can be PO43−, VO43−, SiO43−, or AsO43−; and X can take the form of OH−, Cl−, or F−. HAp is a type of apatite9 and its chemical formula is Ca10(PO4)6(OH)2, where 39% of its weight is Ca, 5.18% is P, and 3.38% is OH. HAp crystallizes in a hexagonal system with a space group of P63/m type; the dimensions of the unit cell are a = b = 9.432 Å, c = 6.881 Å.25 The stoichiometric Ca/P ratio of HAp is 1.666 and its density is 3.219 g cm−3. The structure of HAp consists of an array of tetrahedral phosphates (PO43) constituting the “backbone” of the unit cell; 2 O2 molecules are aligned with axis C, and the other 2 are in a horizontal plane.25–27 Within the unit cell, phosphates are divided into two layers of 1/4 and 3/4 heights, which favor the formation of two types of channels along axis C, referred to as type A and type B, respectively.28
HAp can be synthesized by various techniques using different reactants and production routes, that can modify its physicochemical properties, morphology, chemical composition homogeneity, particle sizes, and degree of crystallinity.5 Daubrée, in 1851, was the first who synthesized HAp by passing phosphorus trichloride vapor on red hot lime.29–32 Since then, various methods of synthesis have been employed:33–37 solid state synthesis, synthesis in aqueous phase,38 growth from molten salts, growth in gel, hydrolysis, mechano-chemical, and the hydrothermal method. In the development of this work was employed the hydrothermal technique because it promotes the growth of various crystals, is cheap and simple to implement. In this method, water is used as a solvent at higher energies to accelerate synthesis and to achieve a better balance of the reaction; this means that products with high crystallinity are obtained, and the corrosion and degradation processes are accelerated.39 These features facilitate the growth of fine crystals, which are homogenous in size, shape, and composition. The growing interest in the hydrothermal technique is derived from its numerous advantages, including the high reactivity of the reactants, easy control of the solution, formation of both the meta-stable phases and the unique phases, less air pollution in the reaction, and low power consumption.40
On Mexican beaches a source of environmental pollution are shells from corals, cockle, and in particular from starfish. These shells due to its high content on CaCO3 can be used as a potential source of the calcium required to produce HAp.41–47 Thus, employing starfish as precursor of HAp, not only generates an important biomaterial, but also provides a solution to deal with this ecological waste.
In this work, HAp synthesis is done by a hydrothermal method using β-tricalcium phosphate (β-TCP) and CaO (obtained from the decomposition of CaCO3 of the starfish of the species “Mellita eduardobarrosoi sp. nov.”). Different working conditions were tested, a ratio of 3:1; for temperatures of 230 °C and 250 °C; and reaction times of 20 and 48 hours. The characterization of the precursors and the obtained products was done through Low Vacuum Scanning Electron Microscopy (LV-SEM), elemental chemical analysis (EDS), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR); which allowed the identification of crystalline phases, morphology, chemical composition, and molecular arrangements. Using a waste product, low cost, short term reactions, close to room temperatures, simplicity of the method and good quality of the HAp produced are some of the advantages of the methodology used in this work, that can be implemented for HAp synthesis at industrial scale.
(1) |
The synthesis process involves mixing in 100 mL of deionized water; 4.5 g of β-TCP, and 0.28 g of CaO of the starfish, which was heat treated. Following this, there was a wait time for the chemical reaction to be effected, which is represented by:
(2) |
The mixture that was obtained was treated under hydrothermal conditions in an autoclave “Cortest Hastolly”, the conditions are shown in Table 1. Here is reported the synthesis made through the hydrothermal method during 20 h and 48 h. The morphology definition and fiber density is larger for longer reaction times as was shown in previous published papers.17,18,50–53
Synthesis number | Temperature (°C) | Pressure (MPa) | Reaction time (hours) |
---|---|---|---|
1 | 230 | 3.4 | 20 |
2 | 250 | 3.4 | 20 |
3 | 230 | 3.4 | 48 |
4 | 250 | 3.4 | 48 |
(a) X-ray diffractometer, XRD, for powders (SIEMENS, model D-5000) with an incident beam wavelength of λCu = 1.5405 Å. Said system was adapted with the JCPDS-ICOD database (International Center of Diffraction Data) version 2.16, which was used for phase identification. The working conditions were as follows: 35 kV; 25 mA; with a sweep carried out in the range of 2.5° to 70° in 2θ with a time of 1 s and an increment of 0.005. The different samples were ground in an agate mortar and they were then placed in respective supports to finally be placed in the diffractometer.
(b) LV-SEM (JEOL model JMS 5900-LV) equipped with a probe for elemental chemical analysis, EDS (OXFORD brand). The operating conditions were as follows: a low vacuum mode; an accelerating voltage of 25 kV; a 75 mA emission current; a working distance (WD) of 10 mm; a pressure of 20 Pa; and a spot size of 40–50 mm. On a 10 mm diameter aluminum cylinder, a carbon ribbon was adhered and the test sample was placed in it.
(c) Fourier transform infrared spectrometer (Fourier transform model NEXUS 670 FT-IR ESP, Niclet brand). Each test sample was previously dried at a temperature of 100 °C for 24 hours. Following this, each sample was mixed with potassium bromide (KBr) for final placement in the spectrometer's chamber.
Fig. 2 Starfish Mellita eduardobarrosoi sp. nov. (a) bottom and top superficial view, (b) superficial morphology, and (c) internal structure. |
Element | Starfish without treatment | Starfish after thermal treatment at 900 °C for 4 h |
---|---|---|
O | 46.50 | 26.05 |
C | 25.98 | 3.01 |
Ca | 24.48 | 65.34 |
Mg | 1.93 | 2.37 |
Al | 0.47 | — |
Si | 0.36 | — |
S | 0.21 | — |
After the grinding process, the morphology of the starfish powder changes, the porous structure has totally disappeared, with particles ranging from 2.5 μm to 22.5 μm (see LV-SEM image, left panel of Fig. 3). Starfish powder after the grinding and cleaning process was also characterized by XRD (upper panel of Fig. 4). Magnesium calcite (CaMg)CO3 is the only phase identified. FTIR spectrum is shown in the bottom of Fig. 4. It can be identified the characteristic peaks of calcite phase localized at 1800 cm−1, 1422 cm−1, 1084 cm−1, 876 cm−1, and 710 cm−1.
Fig. 3 LV-SEM image of the starfish after the grinding process (left panel) and after the thermal treatment (right panel). |
Starfish product after the thermal treatment was also characterized by XRD (Fig. 5 upper panel). Three phases can be appreciated: calcium oxide [CaO], magnesium oxide [MgO], and portlandite [Ca(OH)2]. The latter could be formed after the treatment when it comes into contact with the ambient humidity according to the following chemical reaction:
CaO + H2O → Ca(OH)2. |
Fig. 5 Characterization of the starfish Mellita eduardobarrosoi sp. nov. after the thermal treatment: diffractogram (up) showing the phases: †[Ca(OH)2], ♦[CaO], Θ[MgO], and FTIR spectrogram (bottom). |
FTIR spectroscopy of the starfish after thermal treatment (Fig. 5 bottom panel) indicates the presence of a band at 3641 cm−1, which corresponds to a O–H type bond, associated with the presence of the hydroxyl of the portlandite phase. The bands identified at 876 cm−1, 1120 cm−1, 1420 cm−1 correspond to the calcite phase.
Fig. 8 LV-SEM micrographs showing the products obtained in the synthesis of HAp by hydrothermal process at (a) 230 °C for 20 h, (b) 250 °C for 20 h, (c) 230 °C for 48 h and (d) 250 °C for 48 h. |
Sample | Synthesis 1: 230 °C for 20 h | Synthesis 2: 250 °C for 20 h | Synthesis 3: 230 °C for 48 h | Synthesis 4: 250 °C for 48 h |
---|---|---|---|---|
O | 56.47 | 27.92 | 56.98 | 54.37 |
49.11 | ||||
Ca | 21.64 | 31.91 | 22.21 | 20.59 |
19.94 | ||||
P | 13.58 | 15.72 | 13.39 | 12.98 |
11.57 | ||||
C | 7.97 | 24.44 | 7.03 | 11.24 |
19.20 | ||||
Mg | 0.34 | — | 0.37 | 0.27 |
Ca/P | 1.58 | 2.03 | 1.66 | 1.59 |
1.72 |
Fig. 8b is the micrograph of the HAp sample obtained via hydrothermal technique at 250 °C for 20 hours. Here, it is possible to note greater homogeneity among the abundance of fiber structures. There is a tendency of forming very long fibers with sizes ranging from 36.27 μm to 43.13 μm. Fibers of shorter lengths are still identified, with diameters from 0.98 μm to 1.17 μm. Furthermore, there are smaller structures in the form of cluster of grains with grain sizes ranging from 1.96 μm to 5.88 μm. The morphologies associated with grains are illustrated in the area marked “I” in Fig. 8b, with dimensions of 15.68 μm. The sample is composed of O, Ca, P, and C with minor amounts of Mg and a Ca/P ratio of 1.58 (see Table 3).
In Fig. 8c, the micrograph of the sample obtained at 230 °C for 48 hours is shown. Under these conditions, it can be observed that fiber formation is favored; fibers of different dimensions, which are divided into three groups, are observed. The first group of fibers (I) have diameters of 1.29 μm and average lengths ranging from 12.19 μm to 14.19 μm, the second group of fibers (II) shows lengths ranging from 11.12 μm to 16.12 μm and diameters from 0.64 μm to 0.96 μm and, finally, in the third group (III), which is the most abundant, we can identify fibers with lengths ranging from 7.90 μm to 10.12 μm with an average diameter of 0.32 μm. In the elemental analysis presented in Table 3, it can be seen that, on average, the fibers are composed of O, Ca, P, and C; with a Ca/P ratio of 1.59.
Finally, in Fig. 8d, the LV-SEM micrograph of the sample obtained via the hydrothermal method at 250 °C for 48 hours is observed. The increase in both temperature and time permit the formation of fibers with bigger dimensions (in terms of length and diameter) and with greater homogeneity. It is appreciated that the fibers correspond to the predominant morphology. They are also isolated small particles of sizes ranging from 6.86 μm to 11.76 μm. The fibers have lengths ranging from 41.17 μm to 41.76 μm, diameters varying from 1.96 μm to 2.15 μm. Samples are composed of O, Ca, P, and C, and minor amounts of Mg, with a Ca/P ratio of 1.65 (see Table 3).
For a reaction time of 48 hours, Fig. 9c presents the associated diffraction patterns of the sample synthesized at 230 °C; these patterns are identified only as phases of β-TCP and HAp, where the latter is observed with higher intensity. In the diffractogram of Fig. 9d, the phases of β-TCP and HAp, which were identified after synthesis at a temperature of 250 °C, are observed with a greater signal strength associated to the HAp phase.
Fig. 10 FTIR spectra obtained at: (a) 230 °C for 20 h, (b) 250 °C for 20 h (c) 230 °C for 48 h and (d) 250 °C for 48 h. |
Bond type | Synthesis 1: 230 °C for 20 h | Synthesis 2: 250 °C for 20 h | Synthesis 3: 230 °C for 48 h | Synthesis 4: 250 °C for 48 h |
---|---|---|---|---|
PO (HAp) | 2079 | 2079 | 2079 | |
2002 | 2002 | 2002 | ||
1093 | 1093 | 1093 | ||
1062 | 1062 | 1062 | 1062 | |
1036 | 1036 | 1036 | 1036 | |
964 | 964 | 964 | 964 | |
605 | 605 | 605 | 605 | |
564 | 564 | 564 | 564 | |
471 | 471 | 471 | ||
OH (HAp) | 3568 | 3568 | 3568 | 3568 |
631 | 631 | |||
PO (TCP) | 910 | 910 | 910 | 910 |
716 | 716 | 716 | 716 | |
OH Ca(OH)2 | 3655 | 3655 | 3655 | 3655 |
OH humidity | 3640 | 3640 | 3640 | 3640 |
XRD analysis served to identify the effect of temperature and reaction time on the synthesized HAp. The proportion of the HAp phase tends to increase as the temperature and reaction times growth. This is corroborated by LV-SEM characterization, where HAp homogeneous fibers were identified with a Ca/P ratio close to the stoichiometric when the reaction is carried out at high temperatures and longer reaction times.
Analysis performed by FTIR spectroscopy allowed the identification of absorption bands related to the functional groups of PO4 and OH, as well as the characteristics of HAp and its precursors employed during synthesis, which also validates the results obtained by XRD.
From the set of results obtained in this research, it can be established that the best synthesis conditions for HAp are a temperature of 250 °C during a reaction time of 48 hours. These parameters yielded the best characteristics of HAp, including a more uniform size, greater amounts, homogeneity with a Ca/P ratio near stoichiometric, a higher crystalline degree, and a greater fiber proportion resulting in a ratio of 3:1 of β-TCP and CaO. By forming a large proportion of fibers with very homogeneous lengths and thickness, it was also possible to achieve a Ca/P ratio of 1.65.
Previous works49 used the same starfish Mellita eduardobarrosoi sp. nov., but the monetite (CaHPO4) as a source of PO43−. There the synthesis is carried out at longer reaction times, generating a low density of fibers with diameters from 1.1 μm to 3.3 μm, and 36.1 μm long, and a Ca/P ratio of 1.24 to 1.50. As in the present method, were obtained portlandite products [Ca(OH)2], calcium oxide [CaO] and magnesium oxide [MgO].49 In contrast, when β-TCP is used (as in this paper) longer reaction times (250 hours) promote the formation of a higher density of fibers having sizes ranging from 2.80 μm to 42.10 μm in length and of diameter from 0.60 μm to 1.60 μm, with a Ca/P ratio of 1.65 (practically stoichiometry), and with a higher homogeneity of the fibers obtained, thus producing a better material for biomedical applications. In past research,47 it has been synthetized hydroxyapatite by the solid state method using a starfish and the β-TCP, producing a porous structure with a Ca/P rate of 1.67 (almost stoichiometry). However, the solid state method is expensive and complicated in comparison with the one proposed here. From the previous papers47,49 and the present methodology proposed here, it is clear than the morphology depends not only on the synthesis technique, but also on the reaction times, temperatures, precursor types and concentrations.
The results indicate that the hydrothermal method encourages the formation of HAp with specific characteristics such as dimensions that maintain high homogeneity, a suitable proportion of that phase, and a Ca/P ratio near to stoichiometric. It is also noted that the increase in temperature and reaction time contributes to the improved the amount of HAp phase produced. This study determined that the best condition for the formation of HAp is achieved at a temperature of 250 °C using a 3:1 ratio of β-TCP and CaO, with a reaction time of 48 hours, which promotes the formation of fibers with homogeneous lengths and thicknesses.
The Ca/P ratio of HAp synthesized by the hydrothermal method was 1.65, which is very close to the stoichiometric. From the results obtained by FTIR spectrometry, absorption bands related to the functional groups PO4 and OH, which are characteristic of synthetic HAp were identified. Moreover, bands corresponding to the PO4 ion coming from the β-TCP precursor were also noted. Finally, a band corresponding to the OH bond was identified, which belongs to the Ca(OH)2 phase and could not be detected by XRD, likely because this phase was present in very low proportions.
Success in the production of HAp from starfish and β-TCP through hydrothermal technique suggested that it is possible to produce in a simple, cheap and reproducible method a material that can be used in diverse biotechnological applications.
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