Hydrothermal Synthesis of Hydroxyapatite with Different Morphologies

Article pubs.acs.org/crystal

Hydrothermal Synthesis of Hydroxyapatite with Different Morphologies: Influence of Supersaturation of the Reaction System Yushi Yang,†,§ Qingzhi Wu,†,§ Min Wang,† Jia Long,† Zhou Mao,† and Xiaohui Chen*,‡ †

State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, and Biomedical Materials and Engineering Center of Hubei Province, Wuhan University of Technology, Wuhan, 430070, P. R. China ‡ Department of Prosthetic, School of Stomatology, Wuhan University, Wuhan 430079, P. R. China S Supporting Information *

ABSTRACT: In the present study, hydroxyapatite (HA, Ca5(PO4)3OH) with different morphologies, such as nanorods, microspheres, hexagonal prisms, and hollow flowerlike structure, were synthesized via a facile hydrothermal route by adjusting reaction parameters. The as-synthesized samples were characterized by X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy, and high resolution transmission electron microscopy. Furthermore, the saturation index of the reaction systems under different conditions was approximately calculated in order to explore the formation mechanism of HA. The results indicate that both the saturation index and the intermediates presented at the initial stage of the reaction play crucial roles in the formation of HA with different morphologies. These results provide a promising strategy for the tunable synthesis of HA and other nanomaterials.

1. INTRODUCTION Hydroxyapatite (HA, Ca5(PO4)3OH) is the most stable phase among various calcium phosphates under neutral or basic conditions and is one of the primary components in hard tissues (such as bones and teeth).1 In particular, HA nanostructures with excellent biocompatibility have displayed promising potential in biomedical fields, such as artificial bone,2−4 drug delivery carrier,5−7 anticancer and antibacterial reagents,8−12 cellular imaging,13−15 and so on. Therefore, a great flourish of interest has been focused on the tunable synthesis of HA nanostructures (such as nanoparticles, nanorods, nanowires, nanoflakes, hollow microspheres, flowerlike structures, etc.). Thus far, various strategies have been developed for the tunable synthesis of HA nanostructures, which include hydrothermal route,16−18 coprecipitation,19−21 electrodeposition,22,23and so on. HA is usually synthesized using calcium hydroxide or calclium salts and orthophosphate acid or orthophosphate salts, in aqueous or gel.16−25 In addition, tripolyphosphate (TPP) acid and salt were also utilized as the phosphate source to synthesize HA.26 In this case, calcium ions and TPP ions forms different kinds of complexes dependent on the Ca/P molar ratio,27−30 which could be transformed into HA under hydrothermal treatment through a dissolution−recrystallization process.26 The formation mechanism of HA in aqueous solution is very complicated. In the case of orthophosphate salt/acid used as the phosphate source, amorphous calcium phosphate (ACP) is preferentially formed as a transient phase at the initial stage.31 ACP subsequently converted to other phases, such as dibasic calcium phosphate dihydrate (DCPD, CaHPO4·2H2O), trical© 2014 American Chemical Society

cium phosphate (TCP, Ca3(PO4)2), octacalcium phosphate (OCP, Ca8H2(PO4)6·5H2O), and HA.32−34 Various factors can greatly influence the transformation and crystallization of calcium phosphates, including the biomolecules, pH value of microenvironment, and ion concentrations, among others.35,36 In the case of TPP used as the phosphate source, the phase transformation is much more complicated because the hydrolysis of TPP has to be taken into consideration. However, few efforts have ever been focused on such a process. In this study, calcium chloride (CaCl2) and sodium tripolyphosphate (STPP, Na5P3O10) were used to synthesize Ca-TPP precursor, which was subsequently treated under hydrothermal conditions and resulted in the formation of HA crystals with different morphologies, such as nanorods, microspheres, hexagonal prisms and hollow flowerlike structures. In addition, urea (CO(NH2)2) and glutamic acid were added into the reaction system to adjust the pH value during the synthesis process. The as-synthesized samples were characterized through X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), field-emission scanning electron microscopy (FESEM), and high resolution transmission electron microscopy (HRTEM). Furthermore, the supersaturation degree of the reaction system under different conditions was calculated approximately. These results indicate that both the saturation index and the intermediates presented at a shorter reaction time play crucial roles in the formation of HA with different morphologies. Received: July 15, 2014 Published: August 14, 2014 4864

dx.doi.org/10.1021/cg501063j | Cryst. Growth Des. 2014, 14, 4864−4871

Crystal Growth & Design

Article

2. EXPERIMENTAL SECTION Materials. Sodium tripolyphosphate (Na5P3O10, STTP), CaCl2, and urea ((NH2)2CO) were of analytical grade (Sinopharm Chemical Reagent Co., Ltd.). L-Glutamic acid (Glu) was of biochemical grade (Huashun Biochemical Reagent Co., Ltd.). All reagents were used as received without further purification. Deionized water (16 MΩ·cm) was obtained from a Nanopure Water Systems UV (Thomas Scientific, Swedesboro, NJ). Synthesis of HA Hierarchical Architectures. In a typical synthesis, CaCl2 (0.0555 g, 0.5 mmol) was dissolved in 20 mL of deionized water, Glu (0.2943 g, 2 mmol) was added and dissolved in CaCl2 solution under magnetic stirring with a slight heating. STTP (0.0368 g, 0.1 mmol) was dissolved in another 20 mL of deionized water under magnetic stirring; urea (0.1502 g, 2.5 mmol) was added and dissolved in TPP solution. Then, the solution containing CaCl2 and Glu was added dropwise into the solution containing TPP and urea under magnetic stirring. After 30 min of stirring, the mixture was transferred to and sealed in a 50 mL Teflon-lined stainless steel autoclave and heated to 180 °C for 10 h and then cooled to room temperature. The precipitate was collected and washed alternately with ethanol and deionized water by centrifugation (9000 rpm, 5 min), and then dried at 60 °C in the air. The pH values of the mixture before and after the synthesis reaction were measured by a pH meter. In a series of syntheses, the different molar ratios of reactants were adjusted as listed in Table 1.

Figure 1. FESEM images of the sample S3 (the Ca/P molar ratio of 1.67, the concentration of CaCl2, STTP, Glu, and urea at 12.5, 2.5, 50, and 62.5 mM) synthesized at different reaction times. (a, b) 40 min (c, d) 60 min, (e, f) 600 min.

formed in the center of the flowerlike structures. Figure 1f shows that the central hole was several microns in diameter. Figure 2 shows the FESEM images of P3 synthesized at different reaction times. As shown in Figure 2a,b, a spherical

Table 1. Concentration of Reagents Used for the Synthesis of HA no. b

S0 S1 S2 S3 P0 P1 P2 P3

CaCl2a

STTP

Glu

urea

125 125 125 12.5 125 125 125 12.5

25 25 25 2.5 250 250 250 25

0 250 500 50 0 250 500 50

625 625 625 62.5 625 625 625 62.5

a

The unit of the concentrations is mmol/L. bLetter S in the sample names means Ca/P ratio of the reagent is at stoichiometry of HA (1.67). Letter P in the names means the concentration of phosphate is 10 times higher (Ca/P equals 0.167).

Characterization. The phase structure of the samples was identified by powder X-ray diffraction (XRD) on a D8 Advance diffractometer using Cu Kα radiation (λ = 1.5418 Å). Fourier transform infrared spectroscopy (FT-IR, Nexus, Thermo Nicolet, America) was used to determine the vibration modes characteristic of the samples. The morphology of the samples was observed using fieldemission scanning electron microscopy (FESEM, S-4800, Hitachi Corp, Japan) and high resolution transmission electron microscopy (HRTEM, JEM-2100F STEM/EDS, JEOL Corp, Japan).

Figure 2. FESEM images of the sample P3 (the Ca/P molar ratio of 0.167, the concentration of CaCl2, STTP, Glu, and urea at 12.5, 25, 50, and 62.5 mM) synthesized at different reaction times (a, b) 40 min, (c, d) 60 min, (e, f) 600 min.

structure consisting of nanoplates was obtained when the synthesis was carried out for 40 min. A similar morphology with a larger size was obtained when the hydrothermal reaction was elongated to 60 min, Figure 2c,d. These flowerlike structures vanished when the reaction time was further elongated to 600 min. Instead, a microsized hexagonal prism appeared, as shown in Figure 2e,f. The phase structures of samples S3 and P3 were identified by powder X-ray diffraction (XRD). Figure 3 shows their XRD patterns synthesized at 40, 60, and 600 min, respectively. All of the peaks in the XRD patterns of sample S3 could be assigned to hydroxyapatite (JCPDS 73-0293). Diffraction peaks of sample S3 were sharper when the reaction time was 600 min, suggesting that the longer reaction

3. RESULTS Figure 1 shows the FESEM images of S3 synthesized at different reaction times. As shown in Figure 1a,b, monodispersed microspheres were obtained with an average diameter of ca. 1.3 μm when the synthesis was carried out for 40 min. When the reaction was carried out for 60 min, the flowerlike structures in spherical shape were formed with an average diameter of ca. 14.0 μm, Figure 1c,d. These flowerlike structures consisted of numerous nanoplates. When the reaction time was further elongated to 600 min, the flowerlike structures remained in spherical shape but with a slightly larger diameter (ca. 17 μm), Figure 1e. In particular, an open hole was 4865



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further increased to 4 (S2), a mixture containing microspheres and prismlike structure was obtained, as shown in Figure 4e,f. Figure 5 shows the FESEM image of the samples (P0, P1, and P2) at the reaction time of 600 min. In the absence of Glu

Figure 3. XRD patterns of the sample S3 (the Ca/P molar ratio of 1.67, the concentration of CaCl2, STTP, Glu, and urea at 12.5, 2.5, 50, and 62.5 mM) and P3 (the Ca/P molar ratio of 0.167, the concentration of CaCl2, STTP, Glu, and urea at 12.5, 25, 50, and 62.5 mM) synthesized at different reaction times. (a) S3-40 min; (b) S3-60 min; (c) S3-600 min; (d) P3-40 min; (e) P3-60 min; (f) P3-600 min.

time is favorable to the formation and crystallization of HA. On the other hand, XRD characterization shows that, in the case of P3, HA was obtained when the reaction lasted for 600 min, while other calcium phosphate intermediates instead of HA were obtained in a shorter reaction time of 40 and 60 min. A similar synthesis was carried out by adjusting the reaction parameters for the other samples (S0, S1, S2, and P0, P1, and P2). Figure 4 shows the FESEM images of as-synthesized

Figure 5. FESEM images of the samples (P0, P1, and P2) synthesized under different conditions. (a, b) P0, the Ca/P molar ratio of 0.167, the concentration of CaCl2, STTP, Glu, and urea at 125, 250, 0, and 625 mM, respectively; (c, d) P1, the Ca/P molar ratio of 0.167, the concentration of CaCl2, STTP, Glu, and urea at 125, 250, 250, and 625 mM, respectively; (e, f) P2, the Ca/P molar ratio of 0.167, the concentration of CaCl2, STTP, Glu, and urea at 125, 250, 500, and 625 mM, respectively.

(P0) and when Glu was added into the reaction with Glu/Ca ratio equaled 2 (P1), monodispersed nanorods were obtained as shown in Figure 5c,d. When the synthesis was carried out with Glu/Ca ratio of 4, a prismlike structure was observed in Figure 5e,f. The phase structure of these samples (S0, S1, S2, P0, P1, and P2) was identified by XRD. As shown in Figure 6, all of the peaks in the XRD patterns can be assigned to HA (JCPDS 730293). It is noticeable that the intensity of the diffraction peak derived from the (300) plane was higher than that from the (211) plane in the XRD patterns of S2 and P2, suggesting a preferential growth on the [001] direction.19 This result is in accord with the prismlike structure of the sample S2 and P2. Figure 7 shows TEM and HRTEM images of HA nanorods (S0, P0, and P1) synthesized under different conditions. The nanorods in Figure 7a for S0 were ca. 39.4 nm in length and ca. 18.8 nm in diameter. The interplanar spacing of ca. 0.344 nm obtained from the HRTEM image was ascribed to the adjacent (002) planes of HA crystal. The nanorods in Figure 7c for P0 were ca. 50.9 nm in length and ca. 23.2 nm in diameter. The interplanar spacing of ca. 0.351 nm obtained from the HRTEM image was ascribed to the adjacent (201) planes of HA crystal. The nanorods in Figure 7e for P1 were ca. 54.7 nm in length and ca. 24.4 nm in diameter. The interplanar spacing of ca. 0.344 nm obtained from the HRTEM image was ascribed to the adjacent (002) planes of HA crystal. It is noteworthy that the perfectly aligned lattice planes, as shown in the upper-right insets of Figure 7b,d,f, provide strong evidence for the wellcrystallized nature of nanorods. These different samples (S0, S1, S2, P0, P1, and P2) were also synthesized at a shorter reaction time of 60 min. As shown in Figures S1−S3 (see Supporting Information), a spherical

Figure 4. FESEM images of the samples (S0, S1, and S2) synthesized under different conditions. (a, b) S0, the Ca/P molar ratio of 1.67, the concentration of CaCl2, STTP, Glu, and urea at 125, 25, 0, and 625 mM, respectively; (c, d) S1, the Ca/P molar ratio of 1.67, the concentration of CaCl2, STTP, Glu, and urea at 125, 25, 250, and 625 mM, respectively; (e, f) S2, the Ca/P molar ratio of 1.67, the concentration of CaCl2, STTP, Glu, and urea at 125, 25, 500, and 625 mM, respectively.

samples (S0, S1, and S2) at the reaction time of 600 min. In the absence of Glu (S0), monodispersed nanorods were obtained (Figure 4a,b). When Glu was added into the reaction system with Glu/Ca molar ratio of 2, microspheres (S1) were obtained with a large size distribution (Figure 4c,d). These microspheres consisted of nanoparticles. When the Glu/Ca molar ratio was 4866



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structure was observed in sample S1 and S2. It is interesting that different phases could be indexed to these samples, that is, calcium carbonate for the sample S0, a mixture of calcium carbonate and sodium calcium phosphate for the sample S1, CaHPO4 for the sample S2, HA for the sample P0, and a mixture of calcium pyrophosphate and HA for the sample P1 and P2. The surface structure of the samples synthesized under different conditions was further characterized by FT-IR spectra. Figure 8 shows FT-IR spectra of different samples (S0-S3, P0-

Figure 6. XRD patterns of different samples synthesized under different conditions. (a) S0, the Ca/P molar ratio of 1.67, the concentration of CaCl2, STTP, Glu, and urea at 125, 25, 0, and 625 mM, respectively; (b) S1, the Ca/P molar ratio of 1.67, the concentration of CaCl2, STTP, Glu, and urea at 125, 25, 250, and 625 mM, respectively; (c) S2, the Ca/P molar ratio of 1.67, the concentration of CaCl2, STTP, Glu, and urea at 125, 25, 500, and 625 mM, respectively; (d) P0, the Ca/P molar ratio of 0.167, the concentration of CaCl2, STTP, Glu, and urea at 125, 250, 0, and 625 mM, respectively; (e) P1, the Ca/P molar ratio of 0.167, the concentration of CaCl2, STTP, Glu, and urea at 125, 250, 250, and 625 mM, respectively; (f) P2, the Ca/P molar ratio of 0.167, the concentration of CaCl2, STTP, Glu, and urea at 125, 250, 500, and 625 mM, respectively. The colored vertical lines indicate the diffraction peaks of the (211) and (300) plane.

Figure 8. FT-IR spectra of the samples synthesized at different conditions. (a) S0; (b) S1; (c) S2; (d), S3; (e) P0; (f) P1; (g) P2; (h) P3. The colored vertical lines indicate the wavenumber of different CO32− substitution, red line for type A and blue line for type B.

P3). The absorption bands at ca. 1110, 1030, 962, 604, 565, and 472 cm−1 could be assigned to the vibration of PO43− group.37 The bands at ca. 3570 and 634 cm−1 were assigned to the stretching and liberation vibration of hydroxyl group.37 The wide absorption band at ca. 3440 and 1640 cm−1 was derived from the adsorbed water.9 In addition, the bands at ca. 2924 and 2855 cm−1 could be indexed to the stretching vibration of the −CH2 group contained in Glu molecules, implying the adsorption of Glu molecules on the surfaces of HA nanostructures. It is well-known that the different substitution of CO32− in the HA lattice could be distinguished by FT-IR characterization.37 Namely, A-type substitution of CO32− could be identified by the presence of the bands at ca. 1546, 1465, and 879 cm−1, while B-type substitution of CO32− could be identified by the presence of the bands at ca. 1455, 1415, and 872 cm−1.19 However, it is difficult to differentiate the bands at ca. 1455 and 1465 cm−1, as well as the bands at ca. 872 and 879 cm−1. Therefore, the characteristic band corresponding to the different substitution of CO32− should be observed at ca. 1415 cm−1 for B-type and 1546 cm−1 for A-type. It is obvious that both types of CO32− substitution were observed in FT-IR spectra of most of as-synthesized HA (S0, S1, and S2), and Btype substitution was observed alone in S3, P0, P1, P2, and P3. The intercalation of CO32− into HA lattices might be attributed to the degradation of urea during the synthesis. Therefore, FTIR spectra indicate that all the as-synthesized samples were CO32− substituted HA crystal. In order to obtain HA crystals, TPP ions need to be fully hydrolyzed, giving out free orthophosphate ions that form HA crystal in the solution by interacting with calcium ions and hydroxide ions. The hydrolysis degree could be determined simply by detecting the pH value of the solution. The hydrolysis of TPP could be expressed as follows,

Figure 7. TEM and HRTEM images of HA nanorods synthesized under different conditions. (a, b) S0, the Ca/P molar ratio of 1.67, the concentration of CaCl2, STTP, Glu, and urea at 125, 25, 0, and 625 mM, respectively; (c, d) P0, the Ca/P molar ratio of 0.167, the concentration of CaCl2, STTP, Glu, and urea at 125, 250, 0, and 625 mM, respectively; (e, f) P1, the Ca/P molar ratio of 0.167, the concentration of CaCl2, STTP, Glu, and urea at 125, 250, 250, and 625 mM, respectively. 4867



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5− P3O10 + 2OH− → PO34− + P2O74 − + H 2O

(1)

P2O74 − + 2OH− → 2PO34− + H 2O

(2)

H 2PO−4 ↔ HPO24 − + H+; pK2 = −5.3541 + 0.01984T + 1795.5T −1

HPO24 −

When TPP ions are fully hydrolyzed into orthophosphate, the pH value of the solution could be easily calculated after considering the mass balance, charge balance, activity coefficient, and dissociation balances of orthophosphate acid. STPP solution (25 mM) was incubated at different temperatures (25 °C, 70 °C under water bath, and 180 °C under hydrothermal condition, respectively), and the pH value was measured at different intervals. The pH change of the STPP solution was plotted in Figure 9. It is obvious that STPP

↔

PO34−

+

+ H ; pK3 = 12.023

(6) (7)

In eqs 5 and 6, T stands for temperature in Kelvin, and pK3 is the dissociation constant of H3PO4 at 25 °C. These equations reveal that the concentration and dissociation of H3PO4, H2PO4−, HPO42−, and PO43− depend on the temperature and the pH value of the reaction system. Figure 10 illustrates the

Figure 10. A plot of the distribution of phosphate species against pH value at 180 °C, calculated according to equilibrium eqs 5−7. The colored horizontal lines represent the pH value of different reaction systems before and after the synthesis.

variable distribution of phosphate species (H3PO4, H2PO4−, HPO42−, and PO43−) against the pH value of orthogonal phosphates acids at 180 °C. It is obvious that the dominate phosphate species in the reaction system were H3PO4, H2PO4−, HPO42−, and PO43−, respectively, accompanied by the increase of pH value. Therefore, different calcium phosphate intermediates were formed by adjusting the pH value,40 while the precipitation of HA may occur preferentially under the high pH conditions.41 In the present study, the pH value of the reaction system for different samples was measured before and after the synthesis. The colored horizontal lines shown in Figure 10 represent the measured pH values (data shown in Table S1, Supporting Information). For instance, the pH value of the reaction system for P0 was ca. 7.94 and 9.14 before and after the synthesis. In addition to the relationship between the distribution of phosphate species and pH value, the supersaturation degree of the reaction system may also play important roles in the formation of the different morphologies of HA. The supersaturation degree (S) is defined as42 aa S= A B K sp (8)

Figure 9. pH value of STPP solution at different intervals under different treatments. The red line and orange line show the pH value of STPP solution under a water bath treatment of 20 and 70 °C. The magenta scatter indicates the pH value of STPP solution under hydrothermal treatment of 180 °C. The blue line shows the theoretically calculated pH value of STPP when it is completely hydrolyzed into orthophosphate ions.

solution is stable at room temperature and partly hydrolyzed under a 70 °C water bath even after 12 h. However, TPP ions were fully hydrolyzed under hydrothermal conditions after 60 min. These results indicate that TPP ions were fully hydrolyzed into orthophosphate ions under hydrothermal treatment.

4. DISCUSSION It is well-known that HA is less stable (or better soluble) under acidic conditions (low pH value).1 In this work, STPP was used to gradually release PO43− ions following eqs 1 and 2, while urea was used to adjust the pH value of the reaction system, which was decomposed and resulted in the release of hydroxyl ions during the reaction according to eq 3. HA is usually formed through a precipitation reaction by calcium salts and phosphates gradually, as expressed in eq 4. Equations 5−7 represent the dissociation of H3PO4 and their dissociation constants.36,39 CO(NH 2)2 + 3H 2O → 2OH− + 2NH+4 + CO2

(3)

5Ca 2 + + 3PO34− + OH− → Ca5(PO4 )3 OH

(4)

where aA and aB are the activity of solute A and solute B, Ksp is the solubility constant of the product. In the present study, the supersaturation (S) is given by eq 9 according to eq 4, and the saturation index (SI) is defined as eq 10, ⎛ a 5 2+a 3 3−aOH− ⎞ Ca PO4 ⎟ S = ⎜⎜ ⎟ K sp ⎝ ⎠

⎛ a 5 2+a 3 3−aOH− ⎞ Ca PO4 ⎟ = log (S) SI = log10⎜⎜ ⎟ 10 K sp ⎝ ⎠

H3PO4 ↔ H 2PO−4 + H+; pK1 = −4.5535 + 0.01349T + 799.3T −1

(9)

(10)

The formation of HA nanorod and microprisms could be interpreted according to the calculated SI values under different

(5) 4868



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stable phases like HA.1,46 Some studies also suggested that the crystalline domain might be formed on the surface of ACP and trigger heterogeneous nucleation,47 which lowered the energy barrier for the nucleation and promoted the rate of nucleation.48 Moreover, the crystalline domains formed on the surfaces of ACP were not able to rotate nor relocate, leading to the formation of flowerlike HA structures with a spherical shape.49,50

temperatures and pH values. Using equilibrium constants reported elsewhere,36 the SI value of the samples (S0−S3 and P0−P3) could be calculated with the assistance of PHREEQC (a software capable of dealing with batch reactions).43 In these calculations, STPP and urea were assumed to be fully hydrolyzed into orthophosphate ions, NH4+ and CO32− ions; the effect of Glu on the free energy of HA was ignored.44 Figure 11 shows the calculation results of the SI value for different samples. In these grayscale images, the dark color represents a larger SI value of HA, and the light color represents a smaller SI value. The temperature region is set from 20 to 180 °C, and the pH region is set from 2 to 9. When SI = 0, the reaction between precipitation and dissolution of HA reaches equilibrium. When SI < 0, HA prefers to be dissolved into the solution. When SI > 0, HA is preferentially precipitated from the solution.36 An isothermal line in red was marked in the grayscale maps for the points SI = 0. The vertical lines in different color represent the pH value of the different samples (S1−S3, P1−P3) after the synthesis. The final pH value of the sample S0 and P0 is above 9 and thus were not marked in the grayscale map, but both of them are in the region of SI > 0. As shown in Figure 11, the pH value at 180 °C for the samples (S1−S3 and P1) appeared in the region of SI > 0, implying that HA tended to be precipitated from the reaction system during the synthesis. On the contrary, the pH value at 180 °C for the sample P2 and P3 appeared in the region of SI < 0, suggesting that no HA crystal was precipitated from the reaction system during the synthesis at 180 °C, unless the SI value was above zero due to the decrease of temperature during the cooling process of the synthesis. It is interesting that, in the case of P2 and P3 (Figure 10b,d), the SI value appeared in the region of SI < 0 at 180 °C and in the region of SI > 0 when the temperature was decreased during the cooling process of synthesis. Therefore, it is possible that the prismlike structure was related to the precipitation route. That is, HA nanorods were obtained in the case of SI > 0, while prismlike HA was formed in the case of SI < 0. This assumption is in accordance with the relationship between free energy of HA formation and the temperature, which is defined by −RT ⎛⎜ IP ⎞⎟ −RT ΔG = ln⎜ ⎟ = n ln(S) n ⎝ K sp ⎠

Figure 11. Saturation index (SI) calculated at different temperatures and pH values. The temperature region was set from 20 to 180 °C. The pH region was set from 2 to 9. A darker color indicates a higher value of SI. Red lines indicate the points with the SI value of zero. The pH value of the samples (S1−S3 and P1−P3) was shown as the colored vertical line on the grayscale map. The solution condition in calculation for (a) 125 mM Ca2+, 75 mM PO43−, 250 mM Cl−, 125 mM Na+, 625 mM CO32−, and 1250 mM NH43−; (b) 125 mM Ca2+, 750 mM PO43−, 250 mM Cl−, 1250 mM Na+, 625 mM CO32−, and 1250 mM NH43−; (c) 12.5 mM Ca2+, 7.5 mM PO43−, 25 mM Cl−, 12.5 mM Na+, 62.5 mM CO32−, and 125 NH43−; (d) 12.5 mM Ca2+, 75 mM PO43−, 25 mM Cl−, 125 mM Na+, 62.5 mM CO32−, and 125 mM NH43−.

A similar spherical structure was also observed for the sample S1 and S2 at the reaction time of 60 min (Figure S1, Supporting Information). However, the XRD patterns of these spherical intermediates suggested that a mixture containing different intermediate phases instead of HA were formed at a shorter reaction time (Figure S3, Supporting Information). These spherical intermediate phases were transformed into HA microspheres or prismlike structure when the reaction time was extended to 600 min. It is possible that the growth of HA crystal occurred not only in the period of the reaction (at 180 °C) but also in the period of the cooling process of the synthesis, resulting in the formation of a prismlike structure. By comparison, no spherical intermediates were observed for the other samples (S0, P0, and P1, Figure S2, Supporting Information). Scheme 1 summarized the mechanism responsible for the formation of HA with different morphologies. The final morphology of HA was dependent on both the intermediates formed at the initial stage of reaction (60 min) and the SI value of the reaction system. When spherical HA was formed as the intermediates in the initial stage of reaction, flowerlike HA was obtained (S3). When spherical calcium phosphate intermediates (instead of HA) were formed at the initial stage of reaction,

(11)

in which R is the gas constant, T is the absolute temperature, IP is the ionic activity of the product, S is the supersaturation degree, and n is the number of ions in the formula of HA (n = 9).38 This equation indicates that the absolute value of ΔG decreases as temperature decreases, which renders a slower precipitation rate of HA.45 It is possible that the slow precipitate rate leads to a larger crystal size. The formation of both the flowerlike structure (S3) and spherical structure (S1 and S2) could be attributed to the formation of spherical intermediates during the synthesis. For example, as shown in Figure 1 for the sample S3, HA microspheres were observed (Figure 1a and b) at the initial stage of synthesis (40 min) and subsequently were transformed into a flowerlike structure (Figure 1c,d) after 20 min. This phenomenon could be ascribed to the formation of spherical amorphous calcium phosphate (ACP) and the heterogeneous nucleation of HA on the surface of the former. Previous studies have revealed that ACP usually formed at the early stage of precipitation reaction and subsequently transformed into more 4869



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Natural Science Foundation of China (No. 81190133), the research project of the Health department of Hubei Province for Science and Technology Development (No. 304131958), and the basic research project of Wuhan Science and Technology Bureau (No. 2014060101010041).

Scheme 1. Speculated Mechanism for the Formation of HA Crystals with Different Morphologiesa

■

a The inset FESEM images show HA crystals with different morphologies synthesized at different conditions.

HA microspheres were obtained in the case of SI > 0 (S1 and S2), and prismlike HA was obtained in the case of SI < 0 (P3). When there was no spherical intermediates formed at the initial stage of reaction, HA nanorods were obtained in the case of SI > 0 (S0, P0, and P1), and prismlike HA was obtained in the case of SI < 0 (P2).

5. CONCLUSIONS In summary, HA crystals with different morphologies, including nanorods, hexagonal prisms, microspheres, and flowerlike structures, were successfully synthesized using STPP as the phosphate source via hydrothermal reaction. FT-IR spectra show that CO32− anions were intercalated into HA crystal lattices and resulted in the formation of type A and/or tape B substitution. Approximate calculations on the supersaturation of different reaction systems demonstrate that the SI value of the reaction system, together with the intermediates formed at the initial stage of reaction (60 min), play crucial roles in the formation of HA crystals with different morphologies. The supersaturation-governed formation mechanism of HA crystals not only promotes the understanding on the biomineralization process of bone but also provides an inspiring strategy for the synthesis of various nanomaterials.

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ASSOCIATED CONTENT

S Supporting Information *

FESEM images and XRD patterns of different samples (S0, S1, S2, P0, P1, and P2) synthesized at the reaction time of 60 min pH values of different reaction systems (S0−S3 and P0−P3) before and after the hydrothermal reaction. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86 135 0713 2609. Author Contributions §

These authors contribute equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Natural Science Foundation of China (No. 30800256), the Major Program of 4870



Article

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Hydrothermal Synthesis of Hydroxyapatite with Different Morphologies

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