Synthesis and characterization of hydroxyapatite nanoparticle (PDF ...
Colloids and Surfaces A: Physicochem. Eng. Aspects 322 (2008) 29–33
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Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Synthesis and characterization of hydroxyapatite nanoparticles Burcu Cengiz, Yavuz Gokce, Nuray Yildiz ∗ , Zeki Aktas, Ayla Calimli Department of Chemical Engineering, Faculty of Engineering Ankara University, Ankara 06100, Turkey
a r t i c l e
i n f o
Article history: Received 7 September 2007 Received in revised form 23 January 2008 Accepted 14 February 2008 Available online 21 February 2008 Keywords: Hydroxyapatite Nanoparticle Precipitation method Simulated body fluid CaPTris solution
a b s t r a c t Hydroxyapatite (HA) nanoparticles were synthesized by precipitation method in the presence of simulated body fluid (SBF) solution or calcium phosphor tris (CaPTris) solution which was used for the first time. The synthesized powders were characterized in terms of structure (Fourier transform infrared spectrograph, FTIR and X-ray diffraction, XRD), particle size and morphology (zeta sizer and scanning electron microscopy, SEM). HA crystallites were in hexagonal structure (space group: P63/m). Sizes of crystallites determined at the (0 0 2) and (3 0 0) crystal planes varied from 15.88 to 16.12 nm for CaPTris solution. Although particle size distributions were significantly different for both samples, SEM images of the samples showed that the particles were in nano-sized. Characterization results depicted that HA nanoparticles were also synthesized using CaPTris solution by precipitation method as well as in the presence of SBF solution. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The use of bone-substituted materials in the science of biomaterials is an important objective due to their bioactive and biocompatible properties. Calcium phosphates, major components of natural bone, have bioactive and biocompatible properties. Therefore calcium phosphates have been used in an interdisciplinary field of science involving chemistry, biology, medicine, dentistry and geology for over 20 years. Among calcium phosphate ceramics dicalcium phosphate dihydrate (CaHPO4 ·2H2 O, DCPD), tricalcium phosphate (Ca3 (PO4 )2 , TCP), tetracalcium phosphate (Ca4 P2 O9 , TetTCP), octacalcium phosphate (Ca8 H2 (PO4 )6 , OCP) and hydroxyapatite (Ca10 (PO4 )6 (OH)2 , HA) have been studied for application in medical fields. The only TCP, a resorbable material and HA, a bioactive ceramic, that induces bone formation on its surface, are mainly used as bone-substituted materials [1–4]. HA is not only bioactive but also osteoconductive, non-toxic, non-immunogenic [5]. Although there are many applications of HA such as catalysis, industry of fertilizers and pharmaceutical products, protein chromatography applications, water treatment processes and repairing of bone and teeth, application of pure HA is very limited due to its brittleness. Many efforts have been made to modify HA by polymers since the natural bone is a composite mainly consisted of nano-sized, needle-like HA
∗ Corresponding author. Tel.: +90 312 2126720/1314; fax: +90 312 2121546. E-mail address: [email protected] (N. Yildiz). 0927-7757/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2008.02.011
crystals (accounts for about 65 wt% of bone) [6] and collagen fibers [3,7]. To produce high quality HA bioceramics for artificial bone substitution, ultrafine HA powder was usually employed [8]. In literature, so many methods have been reported for synthesizing HA, including sol–gel [9,10] reverse microemulsion [11,12], hydrothermal [13], microwave-hydrothermal [14], precipitation [6,15] and solid-state reaction [1] methods. Weakly agglomerated and nano-sized HA particles have been synthesized by some of these methods such as hydrothermal and reverse microemulsion methods [8]. The precipitation process is the most reported method for preparing HA particles. This process is simple, low cost and suitable for industrial production but resultant particles have a low quality with a large particle size, wide particle size distribution and a lot of agglomerates [8]. It is known that ultrasonication is particularly effective in breaking up aggregates and in reducing the size and polydispersity of nanoparticles [16] but a few article have studied deagglomeration of HA during the precipitation method. So far, simulated body fluid (SBF) was generally used to synthesize nano-sized HA particles by precipitation method. In this work first time CaPTris solution was used as a different calcium phosphate growth medium. In order to prevent particle agglomeration polyethylene glycol (PEG) was used as a dispersant and also the suspension was treated by ultrasonication. Nanoparticles obtained from the precipitation method using SBF or CaPTris solution were compared in terms of particle sizes and particle size distributions.
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B. Cengiz et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 322 (2008) 29–33
2. Experimental
Table 1 Chemical composition of SBF and CaPTris solutions
2.1. Materials and methods
SBF
2.1.1. Materials The chemicals used were NaCl, NaHCO3 , KCl (99%), MgCl2 (98%), CaCl2 (≥98%), Na2 SO4 , Tris ((CH2 OH)3 CNH2 ) and HA as a reference purchased from Sigma, HCl (37%) and K2 HPO4 obtained from Merck. Polyethylene glycol (Mw = 2000, PEG 2000, Fluka) and ethanol ¨ were also used in the experiments. All reagents (Riedel-de Haen) were used as received. 2.1.2. Preparation of HA nanoparticles with precipitation method using SBF or CaPTris solutions The main calcium phosphate growth medium utilized in our experiments was SBF. SBF [17] was prepared by dissolving NaCl, NaHCO3 , KCl, K2 HPO4 , MgCl2 , CaCl2 and Na2 SO4 in deionized water. Another calcium phosphate growth medium referred to as “CaPTris solution” was prepared by mixing Tris, HCl, K2 HPO4 and CaCl2 in deionized water [18]. The reagents for the two methods (SBF and CaPTris) were added one by one after each reagent was completely dissolved in 1000 and 500 ml deionized water, respectively, in the order given in Table 1 [17,18]. A known amount of CaCl2 (4.4 and 2.2 g) and K2 HPO4 (4.14 and 2.07 g) was added, respectively to the solutions of SBF or CaPTris being stirred to produce the suspension. The suspension was then left still for a day at 37 ◦ C. The resulting suspension was centrifuged and the precipitate was washed with distilled water for five times and then with ethanol for two times. After all washing steps, PEG (1 wt%) and ethanol (precipitate:ethanol = 1:2.5 by volume) were added to the precipitate
CaPTris
Reagent
Amount
Reagent
Amount (g)
NaCl NaHCO3 KCl K2 HPO4 MgCl2 1.0 M HCl CaCl2 Na2 SO4 Tris 1.0 M Cl
8.035 g 0.355 g 0.225 g 0.176 g 0.145 g 39 ml 0.292 g 0.072 g 6.118 g 0–5 ml
Tris ((CH2 OH)3 CNH2 ) HCl K2 HPO4 CaCl2 – – – – – –
24.22 6.570 1.740 2.775 – – – – – –
in the centrifuge tube. After that the suspension in the tube was treated by ultrasonic agitation for 5 min. The resulting precipitate from SBF was dried at 50 ◦ C for 24 h [6]. In the use of CaPTris, the resulting precipitate was dried at 70 ◦ C for 12 h and then sintered at 700 ◦ C for 2 h [8]. The sintered products were crushed using an agate mortar. The crushed powders were treated to ultrasonication for 15 min to reduce the particle size. Ultrasonication was carried out with ultrasonic processor of 20 kHz (SONICS vibra-cell) to 30 kHz (HIELSCHER Ultrasound Technology UP100H) ultrasonic homogenizer. 2.2. Particle characterization Fourier-transformed infrared spectroscopy (FTIR, Model: ALTI Unicam WATTSON 1000) was used in the wave number range of 4000–400 cm−1 . Experimental spectra of solid samples were
Fig. 1. The FTIR spectra of HA samples: (a) reference HA, (b) HA synthesized using CaPTris solution and (c) HA synthesized using SBF solution.
B. Cengiz et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 322 (2008) 29–33
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Table 2 Wave numbers for functional groups of HA samples synthesized from SBF and CaPTris solutions (cm−1 ) Sample
OH−
The water associated
CO3 2−
[13] Reference HA Precipitation from SBF Precipitation from CaPTris
3571 3569 3569 3573
3430 3429 3421 3429
1547 1537 – –
1632 1641 1637 1641
PO4 3− 1457 1457 1461 1457
1415 – – –
1087 1111 1099 1087
1032 1051 1027 1031
961 963 963 961
602 604 600 600
566 576 564 572
471 473 469 469
Fig. 2. XRD patterns of HA: (a) reference and synthesized using CaPTris solution and (b) synthesized using SBF solution.
obtained by preparing KBr plates with a 100:3 ‘KBr-to-HA powders’ ratio. The samples were examined by X-ray diffraction (XRD, Model: Rigaku Dmax 2200 XRD) at the step size of 0.02◦ 2 and the speed of 10◦ 2 per min. A Cu K␣ tube operated at 40 kV and 80 mA was used for the generation of X-rays. The size of the crystallites responsible for the Bragg reflection of the (0 0 2) and (3 0 0) planes was determined using the well-known Sherrer relationship: d=
k , b cos
(1)
˚ k is the shape constant where d is the crystallite diameter in A, ˚ is the Bragg angle in degrees and (∼0.9), is the wavelength in A, b is the observed peak width at half-maximum peak height in rad [12]. Morphology and sizes of the HA powders were investigated by scanning electron microscope (SEM, Model: JEOL JSM-6060LV) and particles size analyser (MALVERN Zetasizer Nano Series Nano-S), respectively. The Ca/P ratio was determined by EDX analysis (Noran System SIX Model 300 X-ray microanalyser). 3. Results and discussion 3.1. FTIR and XRD results The FTIR spectra of HA samples are shown in Fig. 1. The wave numbers of functional groups that belong to HA samples which are synthesized from SBF and CaPTris solutions by precipitation method are given in Table 2. For the reference HA the PO4 3− bands are detected at 473, 576, 604, 963, 1051 and 1111 cm−1 . The water associated with HA is present at 3429 and 1641 cm−1 . The OH− stretching vibration is observed at 3569 cm−1 . The peaks at 1537 and 1457 cm−1 for reference HA, 1461 and 1457 cm−1 for SBF and CaPTris are attributed to the CO3 2− [19] ions which might be the result of the absorption of atmospheric CO2 on the surface of HA particles. These peaks are very weak as seen in the figure obviously this indicates that HA samples obtained from SBF or CaPTris solutions by precipitation method contain very little amount of carbonate [6].
Table 3 Lattice parameters of the synthesized HA samples Sample
Reference HA HA from CaPTris HA from SBF [15]
Lattice parameter (A◦ ) a0
c0
9.7049 9.6993 9.4868 9.4125
6.9427 7.0281 6.8960 6.8765
Unit cell volume (A◦ )3
556.2897 572.5969 537.4800 527.6000
Table 4 Crystallite sizes of the synthesized HA samples Sample
HA from Ref. [12] HA from CaPTris a
Crystallite size for planes, d (nm)a (0 0 2)
(3 0 0)
(0 0 2)/(3 0 0)
26.70 15.88
16.10 16.12
1.66 0.99
From Eq. (1) d = k/(b cos ).
Fig. 2 shows the XRD patterns of reference and the resulting hydroxyapatite samples synthesized from CaPTris (Fig. 2a) and SBF solutions (Fig. 2b). It is reported that nanoparticles of CaPTris was sintered and nanoparticles of SBF was not sintered. The two XRD patterns for the samples (reference and CaPTris) are presented to compare the peak positions and intensities. The XRD pattern of HA particles obtained from CaPTris solution is almost the same with
Fig. 3. Particle size distributions of HA samples synthesized using SBF and CaPTris solution.
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B. Cengiz et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 322 (2008) 29–33
Fig. 4. SEM micrographs of HA samples synthesized by precipitation method: (a) using SBF solution and (b) using CaPTris solution.
Fig. 5. EDX spectra of HA synthesized (a) using SBF solution and (b) using CaPTris solution.
the pattern of HA reference in terms of the peak position and relative intensity. The pattern of HA obtained from SBF solution fit well with the pattern of HA reported in [13,8]. As seen from XRD pattern the sample produced from CaPTris solution was crystalline. The calculated lattice parameters for the unit cell of the hydroxyapatite samples are given in Table 3. Calculated crystallite size values are compared with the values reported in [12] (Table 4). The crystallites varied in a range of very small size from 15.88 to 16.12 nm for the (0 0 2) and (3 0 0) crystal planes, respectively. The crystallite growth was anisotropic in space with the (c) axis direction 0.99 times longer than the (a) axis direction (d0 0 2/d3 0 0). 3.2. Particle size distribution and SEM results Fig. 3 shows that the particle size distribution of HA samples which are synthesized from SBF and CaPTris solutions. As seen in the figure, particle sizes of both samples are quite different. The particle size for the sample synthesized from CaPTris is finer than that of SBF. In terms of particle distribution, the ranges were ∼90–600 and ∼950–2000 nm for the samples produced according to CaPTris and SBF solutions, respectively. SEM micrographs of the HA samples are illustrated in Fig. 4 and the samples possess desultory structure. When the micrographs are examined in terms of particle size, the particle sizes are finer than the particles presented in Fig. 3. This shows that PEG 2000 may not perfectly prevent the agglomeration of very fine particles. SEM
image of HA sample (CaPTris) indicates that the length and width of HA nanoparticles are less than 500 and 100 nm, respectively. Examination of SEM image of HA sample (SBF) clearly shows that the bulk consists of desultory and mainly needle-shaped structures. Needleshaped nanoparticles shown in Fig. 4(a) has a width of 10–50 nm and a length of 120–450 nm. Contradiction of the results obtained from the particle size analyser and SEM is due to particle size analysis method which is based on dynamic light scattering (DLS). The shape of particle strongly affects the angle of scatter, therefore, perfect results are obtained if the particles are spherical. If a suspension contains different shape of particles DLS analysis technique may mislead to interpret the size data. As stated above, it should also be considered the agglomeration level of the particles. The particle size in SEM micrographs is higher than the corresponding crystalite size (Table 4). This discrepancy is observed in the available literature as well [12]. It is estimated that the particles shown in SEM micrographs consist of several crystallites. Therefore the particles are coarser than crystallites. EDX data showed the Ca/P ratio is approximately 1.34 and 1.58 for SBF and CaPTris, respectively. The Ca/P ratio in human bone is 1.67. The Ca/P (1.58) for CaPTris is closer than that of SBF (1.34) comparing with the value of 1.67 (Fig. 5a and b). 4. Conclusion First time HA nanoparticles were synthesized using CaPTris solution as a calcium phosphate medium by precipitation method.
B. Cengiz et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 322 (2008) 29–33
The FTIR spectrum and XRD pattern of HA synthesized using CaPTris solution were fit very well with the reference. The crystallites synthesized in the presence of CaPTris solution varied in a range of very fine size from 15.88 to 16.12 nm. In terms of (d0 0 2/d3 0 0) ratio, which is a measure of uniformity of crystallites, the ratio was 0.99 for the particle synthesized using CaPTris as a medium. As the powder (nanoparticles) produced from SBF contains large fraction of needle-shaped particles, nanoparticles synthesized in the presence of CaPTris medium almost consist of desultory structure. There is an acceptable agreement between DLS and SEM examination results for CaPTris. It may be concluded from examination of the SEM images of HA particles (CaPTris) that the length and width of the particles were less than 500 and 100 nm, respectively. According to EDX analyses, composition of the final product produced from CaPTris (1.58) is closer to the ideal HA (1.67). All findings showed that CaPTris solution as a medium may be suggested to synthesize HA nanoparticles. Acknowledgement The authors thank to TUBITAK, project no. 104M412, for their financial support. References [1] P. Parhi, A. Ramanan, A.R. Ray, A convenient route for the synthesis of hydroxyapatite through a novel microwave-mediated metathesis reaction, Mater. Lett. 58 (2004) 3610–3612. [2] H.R. Ramay, M. Zhang, Preparation of porous hydroxyapatite scaffolds by combination of the gel-casting and polymer sponge methods, Biomaterials 24 (2003) 3293–3302. [3] S. Koutsopoulos, Synthesis and characterization of hydroxyapatite crystals: a review study on the analytical methods, J. Biomed. Res. 62 (2002) 600–612.
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[4] J. Marchi, A.C.S. Dantas, P. Greil, Influence of Mg-substitution on the physicochemical properties of calcium-phosphate powders, Mater. Res. Bull. 42 (2007) 1040–1050. [5] R. Murugan, S. Ramakrishna, Development of nanocomposites for bone grafting, Compos. Sci. Technol. 65 (2005) 2385–2406. [6] F. Wang, M. Li, Y. Lu, Y. Qi, Y. Liu, Synthesis and microstructure of hydroxyapatite nanofibers synthesized at 37 ◦ C, Mater. Chem. Phys. 95 (2006) 145–149. [7] F. Chen, Z. Wang, C. Lin, Preparation and characterization of nano-sized hydroxyapatite particles and hydroxyapatite/chitosan nano-composite for use in biomedical materials, Mater. Lett. 57 (2002) 858–861. [8] F. Xiao, J. Ye, Y. Wang, P. Rao, Deagglomeration of HA during the precipitation synthesis, J. Mater. Sci. 40 (2005) 5439–5442. [9] F. Wang, M. Li, Y. Lu, Y. Qi, A simple sol–gel technique for preparing hydroxyapatite nanopowders, Mater. Lett. 59 (2005) 916–919. [10] I. Kim, P.N. Kumta, Sol–gel synthesis and characterization of nanostructured hydroxyapatite powder, Mater. Sci. Eng. B 111 (2004) 232–236. [11] Y. Sun, G. Guo, Z. Wang, H. Guo, Synthesis of single-crystal HAP nanorods, Ceram. Int. 32 (2006) 951–954. [12] G.C. Koumoulidis, A.P. Katsoulidis, A.K. Ladavos, P.J. Pomonis, C.C. Trapalis, A.T. Sdoukos, T.C. Vaimakis, Preparation of hydroxyapatite via microemulsion route, J. Colloid Interface Sci. 259 (2003) 254–260. [13] Y. Wang, S. Zhang, K. Wei, N. Zhao, J. Chen, X. Wang, Hydrothermal synthesis of hydroxyapatite nanopowders using cationic surfactant as a template, Mater. Lett. 60 (2006) 1484–1487. [14] J. Han, H. Song, F. Saito, B. Lee, Synthesis of high purity nano-sized hydroxyapatite powder by microwave-hydrothermal method, Mater. Chem. Phys. 99 (2006) 235–239. [15] A.C. Tas, Synthesis of biomimetic Ca-hydroxyapatite powders at 37 ◦ C in synthetic body fluids, Biomaterials 21 (2000) 1429–1438. [16] E.S.K. Tang, M. Huang, L.Y. Lim, Ultrasonication of chitosan and chitosan nanoparticles, Int. J. Pharm. 265 (2003) 103–114. [17] T. Kokubo, H. Takadama, How useful is SBF in predicting in vivo bone bioactivity? Biomaterials 27 (2006) 2907–2915. [18] M.R. Mucalo, Y. Yokogawa, T. Suzuki, Y. Kawamoto, F. Nagata, K. Nishizawa, Further studies of calcium phosphate growth on phosphorylated cotton fibers, J. Mater. Sci: Mater. Med. 6 (1995) 658–669. [19] T.A. Kuriakose, S.N. Kalkura, M. Palanichamy, D. Arivuoli, K. Dierks, G. Bocelli, C. Betzel, Synthesis of stoichiometric nanocrystalline hydroxyapatite by ethanolbased sol–gel technique at low temperature, J. Cryst. Growth 263 (2004) 517–523.
Contents lists available at ScienceDirect
Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Synthesis and characterization of hydroxyapatite nanoparticles Burcu Cengiz, Yavuz Gokce, Nuray Yildiz ∗ , Zeki Aktas, Ayla Calimli Department of Chemical Engineering, Faculty of Engineering Ankara University, Ankara 06100, Turkey
a r t i c l e
i n f o
Article history: Received 7 September 2007 Received in revised form 23 January 2008 Accepted 14 February 2008 Available online 21 February 2008 Keywords: Hydroxyapatite Nanoparticle Precipitation method Simulated body fluid CaPTris solution
a b s t r a c t Hydroxyapatite (HA) nanoparticles were synthesized by precipitation method in the presence of simulated body fluid (SBF) solution or calcium phosphor tris (CaPTris) solution which was used for the first time. The synthesized powders were characterized in terms of structure (Fourier transform infrared spectrograph, FTIR and X-ray diffraction, XRD), particle size and morphology (zeta sizer and scanning electron microscopy, SEM). HA crystallites were in hexagonal structure (space group: P63/m). Sizes of crystallites determined at the (0 0 2) and (3 0 0) crystal planes varied from 15.88 to 16.12 nm for CaPTris solution. Although particle size distributions were significantly different for both samples, SEM images of the samples showed that the particles were in nano-sized. Characterization results depicted that HA nanoparticles were also synthesized using CaPTris solution by precipitation method as well as in the presence of SBF solution. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The use of bone-substituted materials in the science of biomaterials is an important objective due to their bioactive and biocompatible properties. Calcium phosphates, major components of natural bone, have bioactive and biocompatible properties. Therefore calcium phosphates have been used in an interdisciplinary field of science involving chemistry, biology, medicine, dentistry and geology for over 20 years. Among calcium phosphate ceramics dicalcium phosphate dihydrate (CaHPO4 ·2H2 O, DCPD), tricalcium phosphate (Ca3 (PO4 )2 , TCP), tetracalcium phosphate (Ca4 P2 O9 , TetTCP), octacalcium phosphate (Ca8 H2 (PO4 )6 , OCP) and hydroxyapatite (Ca10 (PO4 )6 (OH)2 , HA) have been studied for application in medical fields. The only TCP, a resorbable material and HA, a bioactive ceramic, that induces bone formation on its surface, are mainly used as bone-substituted materials [1–4]. HA is not only bioactive but also osteoconductive, non-toxic, non-immunogenic [5]. Although there are many applications of HA such as catalysis, industry of fertilizers and pharmaceutical products, protein chromatography applications, water treatment processes and repairing of bone and teeth, application of pure HA is very limited due to its brittleness. Many efforts have been made to modify HA by polymers since the natural bone is a composite mainly consisted of nano-sized, needle-like HA
∗ Corresponding author. Tel.: +90 312 2126720/1314; fax: +90 312 2121546. E-mail address: [email protected] (N. Yildiz). 0927-7757/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2008.02.011
crystals (accounts for about 65 wt% of bone) [6] and collagen fibers [3,7]. To produce high quality HA bioceramics for artificial bone substitution, ultrafine HA powder was usually employed [8]. In literature, so many methods have been reported for synthesizing HA, including sol–gel [9,10] reverse microemulsion [11,12], hydrothermal [13], microwave-hydrothermal [14], precipitation [6,15] and solid-state reaction [1] methods. Weakly agglomerated and nano-sized HA particles have been synthesized by some of these methods such as hydrothermal and reverse microemulsion methods [8]. The precipitation process is the most reported method for preparing HA particles. This process is simple, low cost and suitable for industrial production but resultant particles have a low quality with a large particle size, wide particle size distribution and a lot of agglomerates [8]. It is known that ultrasonication is particularly effective in breaking up aggregates and in reducing the size and polydispersity of nanoparticles [16] but a few article have studied deagglomeration of HA during the precipitation method. So far, simulated body fluid (SBF) was generally used to synthesize nano-sized HA particles by precipitation method. In this work first time CaPTris solution was used as a different calcium phosphate growth medium. In order to prevent particle agglomeration polyethylene glycol (PEG) was used as a dispersant and also the suspension was treated by ultrasonication. Nanoparticles obtained from the precipitation method using SBF or CaPTris solution were compared in terms of particle sizes and particle size distributions.
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B. Cengiz et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 322 (2008) 29–33
2. Experimental
Table 1 Chemical composition of SBF and CaPTris solutions
2.1. Materials and methods
SBF
2.1.1. Materials The chemicals used were NaCl, NaHCO3 , KCl (99%), MgCl2 (98%), CaCl2 (≥98%), Na2 SO4 , Tris ((CH2 OH)3 CNH2 ) and HA as a reference purchased from Sigma, HCl (37%) and K2 HPO4 obtained from Merck. Polyethylene glycol (Mw = 2000, PEG 2000, Fluka) and ethanol ¨ were also used in the experiments. All reagents (Riedel-de Haen) were used as received. 2.1.2. Preparation of HA nanoparticles with precipitation method using SBF or CaPTris solutions The main calcium phosphate growth medium utilized in our experiments was SBF. SBF [17] was prepared by dissolving NaCl, NaHCO3 , KCl, K2 HPO4 , MgCl2 , CaCl2 and Na2 SO4 in deionized water. Another calcium phosphate growth medium referred to as “CaPTris solution” was prepared by mixing Tris, HCl, K2 HPO4 and CaCl2 in deionized water [18]. The reagents for the two methods (SBF and CaPTris) were added one by one after each reagent was completely dissolved in 1000 and 500 ml deionized water, respectively, in the order given in Table 1 [17,18]. A known amount of CaCl2 (4.4 and 2.2 g) and K2 HPO4 (4.14 and 2.07 g) was added, respectively to the solutions of SBF or CaPTris being stirred to produce the suspension. The suspension was then left still for a day at 37 ◦ C. The resulting suspension was centrifuged and the precipitate was washed with distilled water for five times and then with ethanol for two times. After all washing steps, PEG (1 wt%) and ethanol (precipitate:ethanol = 1:2.5 by volume) were added to the precipitate
CaPTris
Reagent
Amount
Reagent
Amount (g)
NaCl NaHCO3 KCl K2 HPO4 MgCl2 1.0 M HCl CaCl2 Na2 SO4 Tris 1.0 M Cl
8.035 g 0.355 g 0.225 g 0.176 g 0.145 g 39 ml 0.292 g 0.072 g 6.118 g 0–5 ml
Tris ((CH2 OH)3 CNH2 ) HCl K2 HPO4 CaCl2 – – – – – –
24.22 6.570 1.740 2.775 – – – – – –
in the centrifuge tube. After that the suspension in the tube was treated by ultrasonic agitation for 5 min. The resulting precipitate from SBF was dried at 50 ◦ C for 24 h [6]. In the use of CaPTris, the resulting precipitate was dried at 70 ◦ C for 12 h and then sintered at 700 ◦ C for 2 h [8]. The sintered products were crushed using an agate mortar. The crushed powders were treated to ultrasonication for 15 min to reduce the particle size. Ultrasonication was carried out with ultrasonic processor of 20 kHz (SONICS vibra-cell) to 30 kHz (HIELSCHER Ultrasound Technology UP100H) ultrasonic homogenizer. 2.2. Particle characterization Fourier-transformed infrared spectroscopy (FTIR, Model: ALTI Unicam WATTSON 1000) was used in the wave number range of 4000–400 cm−1 . Experimental spectra of solid samples were
Fig. 1. The FTIR spectra of HA samples: (a) reference HA, (b) HA synthesized using CaPTris solution and (c) HA synthesized using SBF solution.
B. Cengiz et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 322 (2008) 29–33
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Table 2 Wave numbers for functional groups of HA samples synthesized from SBF and CaPTris solutions (cm−1 ) Sample
OH−
The water associated
CO3 2−
[13] Reference HA Precipitation from SBF Precipitation from CaPTris
3571 3569 3569 3573
3430 3429 3421 3429
1547 1537 – –
1632 1641 1637 1641
PO4 3− 1457 1457 1461 1457
1415 – – –
1087 1111 1099 1087
1032 1051 1027 1031
961 963 963 961
602 604 600 600
566 576 564 572
471 473 469 469
Fig. 2. XRD patterns of HA: (a) reference and synthesized using CaPTris solution and (b) synthesized using SBF solution.
obtained by preparing KBr plates with a 100:3 ‘KBr-to-HA powders’ ratio. The samples were examined by X-ray diffraction (XRD, Model: Rigaku Dmax 2200 XRD) at the step size of 0.02◦ 2 and the speed of 10◦ 2 per min. A Cu K␣ tube operated at 40 kV and 80 mA was used for the generation of X-rays. The size of the crystallites responsible for the Bragg reflection of the (0 0 2) and (3 0 0) planes was determined using the well-known Sherrer relationship: d=
k , b cos
(1)
˚ k is the shape constant where d is the crystallite diameter in A, ˚ is the Bragg angle in degrees and (∼0.9), is the wavelength in A, b is the observed peak width at half-maximum peak height in rad [12]. Morphology and sizes of the HA powders were investigated by scanning electron microscope (SEM, Model: JEOL JSM-6060LV) and particles size analyser (MALVERN Zetasizer Nano Series Nano-S), respectively. The Ca/P ratio was determined by EDX analysis (Noran System SIX Model 300 X-ray microanalyser). 3. Results and discussion 3.1. FTIR and XRD results The FTIR spectra of HA samples are shown in Fig. 1. The wave numbers of functional groups that belong to HA samples which are synthesized from SBF and CaPTris solutions by precipitation method are given in Table 2. For the reference HA the PO4 3− bands are detected at 473, 576, 604, 963, 1051 and 1111 cm−1 . The water associated with HA is present at 3429 and 1641 cm−1 . The OH− stretching vibration is observed at 3569 cm−1 . The peaks at 1537 and 1457 cm−1 for reference HA, 1461 and 1457 cm−1 for SBF and CaPTris are attributed to the CO3 2− [19] ions which might be the result of the absorption of atmospheric CO2 on the surface of HA particles. These peaks are very weak as seen in the figure obviously this indicates that HA samples obtained from SBF or CaPTris solutions by precipitation method contain very little amount of carbonate [6].
Table 3 Lattice parameters of the synthesized HA samples Sample
Reference HA HA from CaPTris HA from SBF [15]
Lattice parameter (A◦ ) a0
c0
9.7049 9.6993 9.4868 9.4125
6.9427 7.0281 6.8960 6.8765
Unit cell volume (A◦ )3
556.2897 572.5969 537.4800 527.6000
Table 4 Crystallite sizes of the synthesized HA samples Sample
HA from Ref. [12] HA from CaPTris a
Crystallite size for planes, d (nm)a (0 0 2)
(3 0 0)
(0 0 2)/(3 0 0)
26.70 15.88
16.10 16.12
1.66 0.99
From Eq. (1) d = k/(b cos ).
Fig. 2 shows the XRD patterns of reference and the resulting hydroxyapatite samples synthesized from CaPTris (Fig. 2a) and SBF solutions (Fig. 2b). It is reported that nanoparticles of CaPTris was sintered and nanoparticles of SBF was not sintered. The two XRD patterns for the samples (reference and CaPTris) are presented to compare the peak positions and intensities. The XRD pattern of HA particles obtained from CaPTris solution is almost the same with
Fig. 3. Particle size distributions of HA samples synthesized using SBF and CaPTris solution.
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Fig. 4. SEM micrographs of HA samples synthesized by precipitation method: (a) using SBF solution and (b) using CaPTris solution.
Fig. 5. EDX spectra of HA synthesized (a) using SBF solution and (b) using CaPTris solution.
the pattern of HA reference in terms of the peak position and relative intensity. The pattern of HA obtained from SBF solution fit well with the pattern of HA reported in [13,8]. As seen from XRD pattern the sample produced from CaPTris solution was crystalline. The calculated lattice parameters for the unit cell of the hydroxyapatite samples are given in Table 3. Calculated crystallite size values are compared with the values reported in [12] (Table 4). The crystallites varied in a range of very small size from 15.88 to 16.12 nm for the (0 0 2) and (3 0 0) crystal planes, respectively. The crystallite growth was anisotropic in space with the (c) axis direction 0.99 times longer than the (a) axis direction (d0 0 2/d3 0 0). 3.2. Particle size distribution and SEM results Fig. 3 shows that the particle size distribution of HA samples which are synthesized from SBF and CaPTris solutions. As seen in the figure, particle sizes of both samples are quite different. The particle size for the sample synthesized from CaPTris is finer than that of SBF. In terms of particle distribution, the ranges were ∼90–600 and ∼950–2000 nm for the samples produced according to CaPTris and SBF solutions, respectively. SEM micrographs of the HA samples are illustrated in Fig. 4 and the samples possess desultory structure. When the micrographs are examined in terms of particle size, the particle sizes are finer than the particles presented in Fig. 3. This shows that PEG 2000 may not perfectly prevent the agglomeration of very fine particles. SEM
image of HA sample (CaPTris) indicates that the length and width of HA nanoparticles are less than 500 and 100 nm, respectively. Examination of SEM image of HA sample (SBF) clearly shows that the bulk consists of desultory and mainly needle-shaped structures. Needleshaped nanoparticles shown in Fig. 4(a) has a width of 10–50 nm and a length of 120–450 nm. Contradiction of the results obtained from the particle size analyser and SEM is due to particle size analysis method which is based on dynamic light scattering (DLS). The shape of particle strongly affects the angle of scatter, therefore, perfect results are obtained if the particles are spherical. If a suspension contains different shape of particles DLS analysis technique may mislead to interpret the size data. As stated above, it should also be considered the agglomeration level of the particles. The particle size in SEM micrographs is higher than the corresponding crystalite size (Table 4). This discrepancy is observed in the available literature as well [12]. It is estimated that the particles shown in SEM micrographs consist of several crystallites. Therefore the particles are coarser than crystallites. EDX data showed the Ca/P ratio is approximately 1.34 and 1.58 for SBF and CaPTris, respectively. The Ca/P ratio in human bone is 1.67. The Ca/P (1.58) for CaPTris is closer than that of SBF (1.34) comparing with the value of 1.67 (Fig. 5a and b). 4. Conclusion First time HA nanoparticles were synthesized using CaPTris solution as a calcium phosphate medium by precipitation method.
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The FTIR spectrum and XRD pattern of HA synthesized using CaPTris solution were fit very well with the reference. The crystallites synthesized in the presence of CaPTris solution varied in a range of very fine size from 15.88 to 16.12 nm. In terms of (d0 0 2/d3 0 0) ratio, which is a measure of uniformity of crystallites, the ratio was 0.99 for the particle synthesized using CaPTris as a medium. As the powder (nanoparticles) produced from SBF contains large fraction of needle-shaped particles, nanoparticles synthesized in the presence of CaPTris medium almost consist of desultory structure. There is an acceptable agreement between DLS and SEM examination results for CaPTris. It may be concluded from examination of the SEM images of HA particles (CaPTris) that the length and width of the particles were less than 500 and 100 nm, respectively. According to EDX analyses, composition of the final product produced from CaPTris (1.58) is closer to the ideal HA (1.67). All findings showed that CaPTris solution as a medium may be suggested to synthesize HA nanoparticles. Acknowledgement The authors thank to TUBITAK, project no. 104M412, for their financial support. References [1] P. Parhi, A. Ramanan, A.R. Ray, A convenient route for the synthesis of hydroxyapatite through a novel microwave-mediated metathesis reaction, Mater. Lett. 58 (2004) 3610–3612. [2] H.R. Ramay, M. Zhang, Preparation of porous hydroxyapatite scaffolds by combination of the gel-casting and polymer sponge methods, Biomaterials 24 (2003) 3293–3302. [3] S. Koutsopoulos, Synthesis and characterization of hydroxyapatite crystals: a review study on the analytical methods, J. Biomed. Res. 62 (2002) 600–612.
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