1 Introduction

Hydroxyapatite (HAp) Ca10(PO4)6(OH)2 is the most important member of the apatite mineral group. This mineral is the most stable form of calcium phosphate at room temperature and has a pH in the 4–12 range [1–4]. One of the most relevant features of HAp is its presence in the mineralized tissues in the form of microscopic crystals of impure ultra structural complexes (such as enamel). In this case, its crystal size is approximately 1 micron long and a 50 nm diameter. However, in other tissues, such as dentin and bone, the HAp crystal size is smaller than enamel [5]. From the dental standpoint, the crystalline orientation in the enamel is interesting due to the dissolution of the crystals in the process of cavity formation. In this case, the cavity grows faster in the radial direction, parallel to the c-axis of the crystal [6]. Consequently, the properties of HAp are influenced by particle size and morphology. The influence of the preparation methods on the chemical properties of hydroxyapatite has been extensively investigated during the past 20 years, mainly because the HAp is stable in a wide range of compositions, as well as accepting a variety of anionic and cationic substitutions. As a consequence of this, its behavior as biomaterial can be easily modified. The hydroxyapatite texture and morphology are sensitive to the preparation conditions; thus, obtaining hydroxyapatite with desired characteristics could be tailored by appropriate selection of the synthetic pathway [7–10]. A number of methods have been used for HAp synthesis, such as solid-state reactions [11] and mechanochemical synthesis [12, 13]. A very detailed description of the different methods, their differences, synthetic materials used as drying (wet methods) powder methods, etc. can be found in the work published by Sadat-Shojai et al. [14]. From these studies, we learn that the properties of hydroxyapatite powders with particle sizes from the micrometric to the nanometric ranges depend heavily on the preparation method, being the temperature (either during synthesis, drying or the coating process) one of the most relevant parameters [15–17]. Moreover, the hydrothermal synthesis has been broadly defined as a crystallization technology of materials and chemicals directly from aqueous solution by suitable control of thermodynamic (temperature, pressure and chemical composition) and non-thermodynamic variables, such as agitation [18, 19]. For example, Wang et al. [20] has reported that HAp particle size increased with temperature in the range 50–150 °C for a 12–24 h process. The heating time is an important factor for obtaining hydroxyapatite powders with a high crystallinity degree and a Ca/P ratio close to the stoichiometric value [21]. For hydroxyapatite synthesis by microemulsion [22], Zheng et al. [23] have reported the synthesis at a temperature of 85 °C using agarose in a highly alkaline medium, obtaining B-carbonated meso- and macro-porous hydroxyapatite. Besides, works have been reported where microwave radiation is used as a volumetric heating process. For instance, Liu et al. [24] minimized thermal gradients within the reacting materials to obtain hydroxyapatite. Such process is carried out at a temperature of 70 °C for 2 h. There are few reports of hydroxyapatite synthesis at room temperature, for example from Puvvada et al. [1]. However, they heated up the precursors to 80 °C for 1 h to synthesize the material. And natural bones are nanocomposites, consisting of a 69 % of calcium phosphate in the form of crystallized hydroxyapatite and/or amorphous calcium phosphate; the hydroxyapatite crystals have a sheet or needle morphology, and typical dimensions of 20–80 nm [1, 25]. The formation of bone implants based on nanoparticles has the advantageous property that they can be sintered at lower temperatures due to a surface area increase. Furthermore, it is possible to simultaneously improve biological and mechanical performance of bone implants by controlling the characteristics of the powders such as particle shape, particle size, distribution and degree of agglomeration [26].

In this paper, a simple method is presented for hydroxyapatite synthesis by co-precipitation (wet method) at room temperature so at all times we have a chemical structure very close to that of biological hydroxyapatite, this in turn with an eye on for future biomedical applications. To this end, tannic acid was used as stabilizing agent of the HAp nanostructures in water, and two salts such as Ca(NO3)2 and CaCl2 were used; one of our aims was to determine which is best for hydroxyapatite crystal formation at room temperature. The resulting powders were characterized to confirm the presence of HAp. The stoichiometry (molar ratio Ca/P) value was found to be in a range between 1.49 and 1.80 [20].

2 Materials and methods

To synthesize the HAp samples, the following reagents were used: CaCl2·2H2O (Sigma-Aldrich, 99 %), C76H52O46 (Sigma-Aldrich > 99 %), N(C3H7)4OH (Sigma-Aldrich, 25 %), distilled water, H3PO4 (Sigma-Aldrich, 85 %), Ca(NO3)2·4H2O (Sigma-Aldrich, 99 %), Ca5HO13P3 (HAp*,Sigma-Aldrich, 90 %). The HAp synthesis was carried out by means of the co-precipitation method using tannic acid (C76H52O46), tetrapropyl ammonium hydroxide (N(C3H7)4OH), calcium chloride dihydrate (CaCl2·2H2O), calcium nitrate tetrahydrate, Ca(NO3)2·4H2O, phosphoric acid (H3PO4), following the chemical stoichiometry indicated by Puvvada et al. [1]. In this work, the following solutions were prepared: tannic acid 2 %; calcium chloride dihydrate 0.2 M; phosphoric acid 0.12 M; tetrapropyl ammonium hydroxide 0.2 M, and calcium nitrate tetrahydrate 0.2 M. The phosphoric acid was adjusted to pH 9 with tetrapropyl ammonium hydroxide via a gradual process. Once the aqueous solutions were prepared, they were mixed with constant stirring of 300 rpm.

2.1 Samples with CaCl2 salt

The first sample was prepared as follows: the tannic acid and calcium chloride dihydrate solutions were mixed for 20 min. Subsequently, 0.1 mL/min of phosphoric acid was added. As the pH decreases, it is increased with tetrapropyl ammonium hydroxide and allowed to stir for a period of 24 h. In this case, the final pH was 10.

Subsequently, the sample was washed three times with a mixture of methanol and distilled water (1:2) and subjected to a freeze-drying process to obtain a fine powder for further characterization.

2.2 Samples with Ca(NO3)2 salt

Using the same method described above, the synthesis of the second sample was carried out, in which the calcium chloride dihydrate was now replaced by calcium nitrate tetrahydrate, obtaining a final pH 9. Drying is one of the most important stages in the preparation of HAp, because this can strongly affect the particle size. In this paper, the drying is performed by freeze-drying technique to keep the size and structure of the HAp samples [26–28]. In this step, the container containing the final solution is introduced into liquid nitrogen (T = 77 K), to flash-freeze the solution, and then vacuum pumped for 24 h to remove the liquid and obtain samples in solid state.

2.3 Characterization of HAp particles

The synthesized powders were characterized by different techniques. The first analysis was performed by X-ray diffraction (XRD) using a Bruker AXS D8 Advance diffractometer, with CuKα radiation, measuring values of 2θ in a range from 4° to 130° with a step of 0.019°. Subsequently, an analysis of functional groups by Raman spectroscopy was performed using a ThermoScientific confocal microscope with Micro-Raman. The microscope objective was 50×, the laser excitation source had a 10 mW power and a wavelength of 532 nm, with measurements carried out in the range of 3000–50 cm−1, and sample and background exposures were 10 and 12 s, respectively. Finally, a special copper grid (300 mesh) for electron microscopy was prepared for analysis in a JEOL JEM-2010F FasTem transmission electron microscope and in a Hitachi STEM-5500 scanning electron microscope.

3 Results and discussion

The chemical composition of hexagonal HAp (in the P63/m space group) is Ca(I)4Ca(II)6(PO4)6(OH)2. The local atomic configurations are displayed in the schematic representation of Fig. 1a. The Ca(I) site is surrounded by six PO4 3− tetrahedral (Fig. 1b) and coordinated by nine oxygen ions. Moreover, the Ca(II) site is seven-coordinated with six oxygen ions from PO4 3− and one oxygen ion from OH− [29]. In order to determine the effect of tannic acid on the growth of HAp nanostructures, we compare the effect of two different salts in the synthesis method. The HAp precipitation mechanism follows a series of events such as nucleation, aggregation, and growth, where the aggregation plays a highly relevant role on determining the final shape and size of HAp particles [30]. In this synthesis processes tannic acid forms stable complex compounds with Ca2+ ions, especially under acidic conditions, and then treated in the alkaline mixture to provide the alkaline environment for the precipitation of HAp crystals. Then tannic acid plays a significant role in the modification of particle size and morphology.

Fig. 1
figure 1

Crystal structure of HAp viewed along the c-axis

Full size image

3.1 Structural properties

Figure 2 shows the XRD patterns of the HAp samples obtained with the two different calcium salts. Both diffractograms are very similar, and well-defined peaks are observed. Besides that, no other crystalline phase can be identified. The main diffraction peaks are in good agreement with the ICDD, PDF # 01072-1243 card and with those previously reported in other studies using different synthesis methods [31–33]. Indexed peaks correspond to the space group P63/m of the hexagonal phase, which is largely consistent with the work previously reported at room temperature [1]. Diffraction peaks are slightly broadened which indicates small particle size.

Fig. 2
figure 2

XRD patterns of HAp samples with different salts: a Ca(NO3)2 and b CaCl2, respectively

Full size image

Raman vibrations associated with hydroxyapatite present internal tetragonal PO4 3− modes. These are well known, and they have four different vibration modes: ν 1 corresponds to a totally symmetric stretching mode of the tetrahedral PO4 group (P–O bond), ν 2 is a doubly degenerate bending mode of the PO4 group (P–O–P bond), ν 3 is a triply degenerate asymmetric stretching mode of the tetrahedral PO4 group (P-O bond), and ν 4 a triply degenerate bending mode of the PO4 group (O–P–O) [34–36]. Figure 3 shows Raman spectra obtained from the powders. Both spectra are also very similar, and in these cases, three vibration modes can be clearly identified. Figure 3a corresponds to the sample prepared with the Ca(NO3)2 salt. The figure reveals a first band at 1045 cm−1 corresponding to the ν 3 phosphate (PO4) mode. Another appears at 958 cm−1, corresponding to the ν 1 phosphate (PO4) mode, which is the most intensive vibration from HAp, and finally one more is revealed at 583 cm−1, which corresponds to the typical ν 4 band (PO4) of hydroxyapatite [34, 35, 37]. For the sample synthesized with the CaCl2 salt (Fig. 3b), the same bands are identified at slightly different positions 1046, 961, and 584 cm−1, respectively. Both Raman spectra are very close to the pure commercial HAp (see Fig. 3c), which clearly it observed the same number of Raman peaks. These results confirm that hydroxyapatite might be synthesized by this method at room temperature [34].

Fig. 3
figure 3

Raman spectra of HAp samples by using different salts: a) Ca(NO3)2, b) CaCl2 and c) pure commercial HAp*

Full size image

3.2 Morphological properties

SEM analysis showed different particle morphologies for each salt used in the synthesis. Figure 4a, b, reveals hydroxyapatite morphology synthesized with the Ca(NO3)2 salt. It exhibits morphologies of organized flakes less than 100 nm wide. Figure 4c, d shows particles synthesized with CaCl2 salt. In this case, a crystal growth of HAp sheets was promoted with uniform sizes of less than 50 nm. However, unlike those morphologies reported by Wang et al. [28], Cengiz et al. [38] and Zhang [22], our synthesis process was always kept at room temperature.

Fig. 4
figure 4

SEM images of HAp samples synthesized with. a, b Ca(NO3)2 and c, d CaCl2 salt

Full size image

Figure 5 shows TEM images of HAp nanoparticles synthesized with the two different salts. For the case of the Ca(NO3)2 salt, Fig. 5a, b, we show low magnification and high-resolution images, respectively. Here, the morphology and particle size can be identified (see Fig. 5a). Some lattice fringes were observed as well. The inter-planar distance was measured, as shown in Fig. 5b, showing spacings of 0.263 and 0.194 nm, in agreement with the reported spacing of the (202) and (222) planes of HAp structure, respectively. On the other hand, for the synthesis with CaCl2 (Fig. 5c, d), a slight change was observed in particle size and morphology. This indicates a dependency on the salt used for the synthesis. Figure 5d shows the inter-planar distance of a HAp particle, with a spacing of 0.253 nm, which corresponds to the (310) plane [39].

Fig. 5
figure 5

TEM images of HAp samples synthesized with: a, b Ca(NO3)2 and c, d CaCl2 salt

Full size image

Crystal lattice parameters from the analysis of high-resolution images corresponded were found to be in agreement with those reported in the ICDD PDF # 01-072-1243 card. These results are consistent with the observed XRD, as shown in Fig. 2. In the work reported in [14], it is mentioned that the minimum size obtained to date is about 20 nm. Nevertheless, for the synthesis using the two different calcium salts (Fig. 5b, d), some HAp particles obtained by this method were less than 20 nm wide.

In addition, Fig. 6 shows the TEM images of the HAp nanoflakes (Fig. 6a, c) and the corresponding electron diffraction patterns (Fig. 6b, d), indicate clearly visible rings, whose interplanar spacing are in good agreement with the characteristic spacing of HAp structure. The images reveal the polycrystalline nature of the HAp prepared by the co-precipitation method at room temperature using both calcium salts. The (102), (211), (202), and (131) distinctive planes of the HAp phase can be observed [40].

Fig. 6
figure 6

TEM and electron diffraction patterns of HAp samples synthesized with: a, b Ca(NO3)2 and c, d CaCl2

Full size image

In order to confirm the elemental composition and the atomic percentages present in the HAp samples, energy-dispersive spectroscopy (EDS) analysis was performed. Figure 7 shows the spectra obtained for each sample: It can be observed that both samples have similar percentages of the most characteristic hydroxyapatite elements such as Ca and P. Similar EDS spectra of hydroxyapatite nanoparticles deposited on metal surfaces were reported in [41]. Figure 7a, b shows the atomic percentages obtained by EDS; it shows that the samples prepared with Ca(NO3)2 have a Ca/P ratio of 1.54 (at.%), while those prepared with CaCl2 have a ratio of 1.65 (at.%), values are consistent with those previously reported [42]. However, different hydroxyapatite characterization studies [43–45] shown that EDS analysis is not reliable in the identification of CaP phases, but in combination with Raman, XRD, and TEM, it is an effective tool for the identification of Hydroxyapatite. The use of EDS data to obtain a localized chemical qualitative analysis is evident.

Fig. 7
figure 7

EDS of HAp samples synthesized with, a Ca(NO3)2 and b CaCl2 salt

Full size image

4 Conclusions

In conclusion, HAp nanoparticles were synthesized by a co-precipitation method using tannic acid as stabilizing agent. Two different calcium salts were used (Ca (NO3)2 and CaCl2), and in both cases they were obtained in an aqueous solution. Subsequently, they were dried by a freeze-drying technique in order to obtain a powder as a final product. Each powder was characterized by X-ray diffraction, Raman spectroscopy and electron microscopy. The analysis confirms that hydroxyapatite nanoparticles have a hexagonal structure. More important, during the whole synthesis and drying process the samples were required to raise the room temperature. This opens the possibility to develop composite biomaterials for biomedical applications at room temperature or physiological temperature based in hydroxyapatite nanocompounds.