Modification of Calcium Hydroxyapatite Using Alkyl Phosphates

Jan 15, 1997 - After the treatment, only rod-shaped particles were observed, testifying that the surfaces of the original CaHAP particles were modifie...
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Modification of Calcium Hydroxyapatite Using Alkyl Phosphates Hidekazu Tanaka, Akemi Yasukawa, Kazuhiko Kandori, and Tatsuo Ishikawa* School of Chemistry, Osaka University of Education, 4-698-1 Asahigaoka, Kashiwara-shi, Osaka-fu 582, Japan Received April 29, 1996. In Final Form: November 19, 1996X Synthetic colloidal calcium hydroxyapatites (Ca10(PO4)6(OH)2, CaHAP), treated with hexyl, octyl, and decyl phosphates in acetone-water solutions, were characterized by various means. XRD patterns of the modified materials showed a strong peak and two weak ones besides the peaks due to CaHAP. The d value of these three additional peaks linearly increased with an increase in the carbon number of the phosphates. Upon increasing the concentration of the phosphates in the treating solution, these peaks grew, while the peaks due to CaHAP diminished. After the treatment, only rod-shaped particles were observed, testifying that the surfaces of the original CaHAP particles were modified without formation of new particles. These results show that the modified CaHAP particles have a surface-layered phase consisting of multilayers of alternating octacalcium phosphate (OCP)-like phase and bimolecular layer of alkyl groups. Alkyl groups and H2O in the layers were removed by outgassing above 200 °C to give the materials having mesopores.

1. Introduction Synthetic calcium hydroxyapatite, Ca10(PO4)6(OH)2 (abbreviated CaHAP), is a component of the hard tissues of animal organisms. This material finds applications as adsorbents, catalysts, and bioceramics. In these usages, interaction of the CaHAP surface with other substances is a very important factor. So far, we have investigated the surface structure and properties of various hydroxyapatites such as CaHAP,1 strontium hydroxyapatite (SrHAP),2 solid solution of CaHAP and SrHAP,3 silicacoated CaHAP,4 and Fe(III)-substituted CaHAP.5 It has been established that these hydroxyapatites have several kinds of surface P-OH groups acting as adsorption sites for various molecules.6 Hence, modifying the CaHAP surface is expected to control the surface structure and property, leading to giving a novel function to CaHAP. Lebugle et al. have modified CaHAP with (hydroxyethyl)methacrylate phosphate and dodecanol phosphate by a coprecipitation method in order to disperse the CaHAP particles in polymers as a filler cement used in dental and medical therapies.7-9 However, they have not fully clarified the surface structure of the modified CaHAP. Recently, we have studied the structure and properties of the CaHAPs modified with hexyl and decyl phosphates in acetone solution10 and ethyl phosphate in acetone-water solutions.11 Only the surface of the CaHAP was modified * Corresponding author. Fax: +81-729-78-3394. X Abstract published in Advance ACS Abstracts, January 15, 1997. (1) Ishikawa, T.; Wakamura, M.; Kondo, S. Langmuir 1989, 5, 140. (2) Ishikawa, T.; Saito, H.; Kandori, K. J. Chem. Soc., Faraday Trans. 1992, 88, 2937. (3) Ishikawa, T.; Saito, H.; Yasukawa, A.; Kandori, K. J. Chem. Soc., Faraday Trans. 1993, 89, 3821. (4) Ishikawa, T.; Wakamura, M.; Kawase, T.; Kondo, S. Langmuir 1991, 7, 596. (5) Ishikawa, T.; Saito, H.; Yasukawa, A.; Kandori, K. Bull. Chem. Soc. Jpn. 1996, 69, 899. (6) Ishikawa, T.; Kondo, S. In Fundamentals of Adsorption; Mersmann, A. B., Scholl, S. E., Eds.; Engineering Foundation: New York, 1991; p 321. (7) Lebugle, A.; Subirade, M.; Delpech, V. In Hydroxyapatite and Related Materials; Brown, P. W., Constatz, B., Eds.; CRC: London, 1994; p 231. (8) Subirade, M.; Lebugle, A. Ann. Chim. Fr. 1991, 16, 41. (9) Subirade, M.; Lebugle, A. Ann. Chim. Fr. 1993, 18, 183. (10) Tanaka, H.; Yasukawa, A.; Kandori, K.; Ishikawa, T. Colloids Surf., in press. (11) Ishikawa, T.; Tanaka, H.; Yasukawa, A.; Kandori, K. J. Mater. Chem. 1995, 5, 1963.

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with hexyl and decyl phosphates in acetone solution, while the treatment with ethyl phosphate in acetone-water solutions influenced the bulk structure. In the latter case, the modified particles exhibited a layered structure including ethyl groups and H2O. However, the detailed formation mechanism of the layered structure has not been satisfactorily investigated. It is of interest in biological and material sciences to clarify the interaction between CaHAP and alkyl phosphates, which is important not only in controlling the affinity of CaHAP for biomaterials but also in biological mineralization and calcification, because a variety of organic phosphates are contained in animal organisms. Moreover, the modification of CaHAP with alkyl phosphates is anticipated to produce new organic-inorganic composite materials. The present study was conducted to elucidate the structure of the CaHAP modified with alkyl phosphate and the modification mechanism. We treated the synthetic CaHAP particles with acetone-water solutions of hexyl, octyl, and decyl phosphates with longer alkyl groups than ethyl group used in the previous study.11 The modification mechanism is discussed based on the structure of the modified materials. 2. Experimental Section Materials. Synthetic calcium hydroxyapatite particles were prepared by a wet method.12 Ca(OH)2 (0.405 mol) was dissolved into 20 dm3 of deionized-distilled water free from CO2 in a sealed Teflon vessel. After the vessel was stirred for 24 h at room temperature, 0.249 mol of H3PO4 was added to the Ca(OH)2 solution, and the resulting suspensions were stirred for further 24 h at room temperature and then aged at 100 °C for 48 h. The CaHAP particles generated were filtered off, thoroughly washed with distilled-deionized water, and finally dried in an air oven at 70 °C for 16 h. Modification of CaHAP was performed as follows. One gram of CaHAP was treated with 0.01-0.10 mol dm-3 of monohexyl (HP), monooctyl (OP), and monodecyl phosphate (DP) solutions, of which the solvents were a mixture of acetone and water (2:1), by refluxing at the boiling point of the solution (64 °C) for 5 h. The particles thus treated were washed with acetone by ultrasonication and centrifugation and were dried by evacuating at room temperature for 16 h. HP, OP, and DP were synthesized by reacting pyrophosphoric acid with hexanol, octanol, and decanol, respectively.13 All the chemicals used were reagent (12) Bett, J. A. S.; Christner, L. G.; Hall, W. K. J. Am. Chem. Soc. 1967, 89, 5535.

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Figure 1. XRD patterns of CaHAPs modified with various DP concentrations (mol dm-3): unmodified sample (a, a′), 0.01 (b, b′), 0.02 (c, c′), 0.03 (d, d′), 0.05 (e, e′), 0.075 (f, f′), and 0.10 (g, g′). grade. The purity of the synthetic phosphates was certified by FTIR and CHN elemental analysis. Characterization. The CaHAP particles thus treated were characterized by conventional methods. X-ray powder diffraction patterns were taken with a Rigaku diffractometer using Nifiltered Cu KR radiation (30 kV, 15 mA). The Ca2+ and PO43contents of the unmodified materials were measured by a Seiko inductively coupled plasma (ICP) spectrometer and a molybdenum blue method, respectively, by first dissolving in an HCl solution. The carbon contents were determined by a Yanagimoto CHN elemental analyzer. The particle size and shape were observed using a Jeol transmission electron microscope (TEM). Thermal gravimetry (TG) and differential thermal analysis (DTA) were simultaneously measured with a Seiko thermoanalyzer at a heating rate of 5 °C min-1 in an air stream and an N2 stream. The specific surface areas and pore size distribution curves were obtained from N2 adsorption isotherms measured by a volumetric apparatus at the boiling temperature of N2. Before the adsorption, the samples were outgassed at various temperatures from 100 to 400 °C for 2 h. Transmission IR spectra were taken in vacuo at room temperature by a self-supporting disk method using a Digilab Fourier transform near-infrared (FTNIR) spectrometer with a PbSe detector.

3. Results and Discussion Structure Change of CaHAP by Modification. Figure 1 shows the XRD patterns of the CaHAP treated with different concentrations of DP. As seen in this figure, peaks in addition to those from the original CaHAP are seen; particularly, one strong peak and two weak ones appear below 2θ ) 10°. With increasing DP concentration, the new peaks grow without peak shift and the CaHAP peaks diminish; the intensities of the CaHAP peaks of the material modified with 0.10 mol dm-3 DP (pattern g′) are about half of those of unmodified CaHAP (pattern a′). Figure 2 compares the XRD patterns below 2θ ) 15° for CaHAPs treated with 0.10 mol dm-3 HP, OP, and DP. The materials modified with HP and OP also give rise to a strong peak and two weak ones. The positions of these new peaks shift to a low diffraction angle with an increase in the length of the alkyl groups of the phosphates. The assignment of these new peaks will be described in detail below. Figure 3 displays the TEM pictures of the unmodified CaHAP particles and the particles treated with 0.10 mol dm-3 OP and DP. Since no particles are observed except rod-shaped particles, no new particles are generated by (13) Nelson, A. K.; Toy, A. D. F. U.S. Patent No. 3,146,255, Aug 25 1964.

Figure 2. XRD patterns of CaHAPs modified with 0.10 mol dm-3 HP (a), OP (b), and DP (c).

this modification. The particle width increases by the treatment, while the particle length is almost unchanged. Figure 4 illustrates the distribution curves of the particle width obtained from the TEM pictures of the particles treated with different DP concentrations. The particle width increases with increasing DP concentration; the mean particle width increases from 15 to 18 nm with increasing DP concentration from 0 to 0.10 mol dm-3. A similar particle growth was observed in the modification with HP and OP. However, as will be described later, the modified samples were decomposed by a electron beam during the TEM observation. Therefore, Figure 4 does not show the exact sizes of the modified particles but shows the sizes of the decomposed particles. The particle widths of the modified materials are still larger than that of the original CaHAP even after the decomposition. Consequently, we can at least recognize that the modification reaction takes place on the unmodified particles without formation of new kinds of particles, although the precise particle sizes were not determined from the TEM pictures. Thermal Treatment of Modified CaHAP. To examine the thermal stability of the alkyl groups in the modified particles, the modified samples were outgassed at different temperatures from 100 to 400 °C for 2 h. The amount of alkyl groups in the modified materials thus treated was estimated from the carbon content. Figure 5 shows the amount of alkyl groups in the materials treated with 0.10 mol dm-3 OP and DP as a function of outgassing temperature. The amount of alkyl groups linearly decreases between 100 and 300 °C and approximates 0 above 300 °C. The samples treated with OP and DP exhibit an almost identical amount of alkyl groups. When outgassed above 300 °C, three additional XRD peaks in Figure 2 completely disappeared, while the intensities of the peaks due to CaHAP remained unchanged. Therefore, heating above 300 °C changes the crystalline parts produced by the modification to be amorphous. Figure 6 depicts the TG and DTA curves taken in an air stream for the materials treated at different DP

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Figure 3. TEM pictures of the particles unmodified (a) and modified with 0.10 mol dm-3 OP (b) and 0.10 mol dm-3 DP (c).

concentrations by the solid and dotted lines, respectively. TG curve a of the unmodified material shows a continuing weight loss from room temperature to ca. 1000 °C, which is mainly due to the removal of H2O strongly adsorbed on the CaHAP particles as confirmed by FTIR.1 TG curves b-g of the modified materials give rise to a weight loss between 200 and 300 °C besides the monotonous weight loss observed on the untreated material. The weight loss between 200 and 300 °C increases with an increase in the carbon content and is gradually separated into two steps at ca. 200 and 250 °C (named steps 1 and 2, respectively). The total weight loss in these two steps was larger than the mass of the included decyl groups estimated from the carbon contents. Hence, this weight loss should be caused by removal of another substance besides the decyl groups. DTA curves e′-g′ of the modified materials clearly show an endothermic peak at step 1 and an exothermic peak at step 2, though no peak is detected in curve a′ of the unmodified material. However, on increasing the DP concentration, the exothermic peak becomes weak and the endothermic peak develops. For the assignment of these peaks, TG-DTA curves of the modified materials were taken in an N2 stream. Although all the TG curves in an N2 stream were almost the same as the curves measured in an air stream, the exothermic peak at step 2 observed in an air stream turned endothermic. Two

endothermic DTA peaks of steps 1 and 2 detected in an N2 stream became strong and sharp with increasing DP concentration. It seems, therefore, that the exothermic and endothermic peaks observed in an air stream are assigned respectively to combustion of decyl groups and removal of H2O. The existence of H2O in the modified materials was confirmed by FTIR, as described below. The exothermic peak due to combustion of decyl groups is weakened as the DP concentration increases. This may be because the combustion of decyl groups is inhibited by H2O evolved in step 1. The materials treated with HP and OP showed similar TG-DTA curves to those of the DP-modified materials. IR Spectra of Modified CaHAP. We measured the IR spectra to clarify the structure of the modified materials. Figure 7 compares the transmission IR spectra of the materials untreated and treated with DP taken in vacuo at room temperature after outgassing at 200 and 300 °C for 2 h. Spectrum a of the unmodified sample outgassed at 300 °C has a strong band at 3572 cm-1 and three bands at 3680, 3670, and 3655 cm-1. The former 3572-cm-1 band is due to lattice OH- ions, and the latter three bands are assigned to surface P-OH groups resulting from protonation of surface PO43- ions to maintain the surface charge balance of CaHAP,1 whereas spectrum b of the sample treated with 0.05 mol dm-3 DP and outgassed at 200 °C

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Figure 4. Particle width distribution curves of CaHAPs modified with different DP concentrations (mol dm-3): 9, unmodified sample; 0, 0.02; O, 0.05; 4, 0.10.

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Figure 6. TG-DTA curves in air of CaHAPs modified at various DP concentrations. TG and DTA curves are represented by the solid lines (a-g) and the dotted ones (a′-g′), respectively. DP concentration (mol dm-3): unmodified sample (a, a′), 0.01 (b, b′), 0.02 (c, c′), 0.03 (d, d′), 0.05 (e, e′), 0.075 (f, f′), and 0.10 (g, g′).

Figure 7. In vacuo IR spectra of CaHAPs unmodified and modified with 0.05 mol dm-3 DP: (a) unmodified and outgassed at 300 °C, (b) modified and outgassed at 200 °C, (c) modified and outgassed at 300 °C. Figure 5. Plots of the amount of alkyl group in the CaHAPs modified with 0.10 mol dm-3 of OP (O) and DP (4) vs outgassing temperature.

has strong bands at 2960, 2920, and 2855 cm-1 due to decyl groups but no surface P-OH bands. The broad band centered at 3200 cm-1 is stronger than that of spectrum a of the unmodified material. This broad band can be assigned to the H2O strongly adsorbed and/or bound on CaHAP.1 Therefore, the modified material contains more H2O compared to the unmodified one. After outgassing at 300 °C, the bands of the decyl groups almost disappear, a surface P-OH band appears at 3670 cm-1, and the broad band at 3200 cm-1 is reduced (spectrum c). These FTIR results imply that the decyl groups are eliminated by outgassing above 300 °C and that the kind and number of surface P-OH groups generated by the evacuation differ from those of the groups of the unmodified CaHAP. Modification Mechanism. Assuming that alkyl groups exist on the surface of the modified materials, the number of alkyl groups per unit area of the particle surface (denoted na) was estimated from the carbon content using the specific surface area of the original CaHAP. The na

increased from 1.3 to 19.2 groups nm-2 with an increase in DP concentration from 0.01 to 0.10 mol dm-3. The number of decyl groups to cover the particle surface is 5 groups nm-2 as estimated by assuming the cross-sectional area of this group to be 0.2 nm2 and perpendicular orientation to the particle surface in the closest packing. The samples treated with a high DP concentration show larger na than 5 groups nm-2, which reveals that the alkyl phosphates exist not only on the particle surface but also inside the particle. However, from the crystal structure of CaHAP, these phosphates having long alkyl groups are hardly embraced in the CaHAP crystals without crystal distortion. Accordingly, a new phase would be formed on the CaHAP particles by the modification. In a previous paper, we proposed the formation of a layered structure on CaHAP by the modification with ethyl phosphate (EP) in acetone-water solution.11 The layer consists of a phase like octacalcium phosphate, Ca8(HPO4)2(PO4)4‚5H2O (called OCP). The materials modified with HP, OP, and DP are predicated to take structure similar to the EP-modified one. It has been established that OCP is prepared from a saturated calcium ortho-

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Figure 8. Plots of d values of three XRD diffraction peaks of the modified CaHAPs shown in Figure 2 vs carbon number of alkyl groups of phosphates.

phosphate solution at pH ) 6 and 60 °C or pH ) 4 and 70 °C and is composed of a layered structure having alternating “hydrated layer” and “CaHAP layer”.14,15 The treating condition in this study resembles that of the OCP formation. The alkyl phosphate ions that are less acidic compared to the HPO42- ions would offer a suitable pH for the OCP formation. Furthermore, the modification was carried out at 64 °C near to the formation temperature of OCP. Monma has synthesized double salts of OCP and dicarboxylic acids by the hydrolysis of R-Ca3(PO4)2 and found that the d value of the (100) plane of OCP is expanded with increasing carbon number of the dicarboxylic anions.16 It seems reasonable, therefore, to consider that the “OCP-like phase” was formed by modification with HP, OP, and DP. As seen in Figure 1, the XRD patterns of the modified materials show a strong peak at a low diffraction angle. Figure 8 shows the d values of these peaks against the carbon number of the phosphates. The d value increases linearly with an increase in the carbon number, i.e., the length of the alkyl groups, suggesting the possibility of the formation of bimolecular layers of the alkyl phosphates. Yamanaka et al. have found that the zirconium phosphates treated with alcohols take a layered structure containing alkyl phosphates.17 The reported d spacing of the bimolecular layer of the alkyl phosphates formed by treating with alcohols having the same carbon number as the alkyl phosphates used in the present study is close to the d spacing of the strong XRD peak in Figure 2. Hence, the strong peaks can be assigned to the scattering from the (100) plane of the new layered phase formed on the CaHAP particle by treating with alkyl phosphates. Based on the above-mentioned results, we can propose a layered structure of the new phase as illustrated for the sample treated with DP in Figure 9. All the decyl groups are oriented perpendicularly to the surface of OCP-like phase on the CaHAP substrate. The bimolecular layer of the alkyl phosphates is organized between the OCP-like phases due to the hydrophobic property of the alkyl groups. The layered structure made up of an alternating bimolecular layer and OCP-like phase gives a multilayer. The (14) Monma, H.; Ueno, S.; Kanazawa, T. J. Chem. Tech. Biotechnol. 1981, 31, 15. (15) Elliott, J. C. Structure and Chemistry of the Apatites and Other Calcium Orthophosaphates; Elsevier: Amsterdam, 1994; p 12. (16) Monma, H. Bull. Chem. Soc. Jpn. 1984, 57, 599. (17) Yamanaka, S.; Matsunaga, M.; Hattori, M. J. Inorg. Nucl. Chem. 1981, 43, 1343.

Figure 9. Schematic structure of the layered phase on DPmodified CaHAP.

Figure 10. Plots of specific surface areas of CaHAPs unmodified (9) and modified with 0.10 mol dm-3 HP (0), OP (O), and DP (4) vs outgassing temperature.

crystallite size of the layered phase for the material treated with 0.10 mol dm-3, evaluated from the (100) peak at 2θ ) 2.82° shown in Figure 1 using the Scherrer equation, is 37 nm larger than 18.1 nm, the mean particle width estimated from the TEM picture. This discordance could be ascribed to a shrinkage of the modified particles because of the release of alkyl groups during the TEM measure-

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Figure 11. Pore size distribution curves of CaHAPs modified with 0.10 mol dm-3 DP and outgassed at various temperatures for 2 h. The unmodified sample (9) was outgassed at 300 °C, and the modified samples were outgassed at 100 (0), 200 (O), and 300 °C (4).

ment. The alkyl groups were removed by outgassing below 300 °C as shown in Figure 5, and the XRD peaks due to the new phase completely disappeared on outgassing at 300 °C, suggesting the possibility of the removal of alkyl groups by a strong electron beam of the TEM. Actually, the morphology of the modified samples varied during the TEM observation. The exact particle width and the thickness of the new phase were not determined from the TEM pictures. Pore Structure of Modified CaHAP. The alkyl groups and H2O in the modified CaHAP were removed by outgassing at elevated temperatures. To confirm the formation of pores in the outgassed samples, we measured N2 adsorption isotherms of the samples outgassed at various temperatures from 100 to 400 °C for 2 h. Figure 10 shows the BET specific surface areas of the materials untreated and treated with 0.10 mol dm-3 HP, OP, and

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DP as a function of outgassing temperature. The surface area of the unmodified material almost does not vary with elevating the outgassing temperature. On the other hand, the surface areas of the modified materials are increased by outgassing above 200 °C and are larger than that of the unmodified one outgassed at 200 and 300 °C. Figure 11 shows the pore size distribution curves obtained from the N2 adsorption isotherms using the Dollimore-Heal method18 for the materials unmodified and modified with 0.10 mol dm-3 DP. As shown by the solid square symbols, the unmodified material has mesopores with a mean pore diameter of ca. 4 nm which may be openings among the particles. The distribution curve shown by the open circle symbols of the DP-modified sample outgassed at 200 °C has a maximum at a diameter of ca. 2 nm. These mesopores would be due to the removal of alkyl groups and H2O molecules from the layered phase. The t-plot analysis19 of the N2 adsorption isotherms proved that all the modified and unmodified particles have no micropores with a pore diameter < ca. 1 nm. As consequence, the results of XRD and N2 adsorption testify that the layered phases formed on CaHAP turn amorphous and porous upon outgassing at 200 and 300 °C. 4. Conclusions The multilayers consisting of bimolecular alkyl phosphate layers and OCP-like phases were formed on the CaHAP substrate by the treatment with alkyl phosphates in acetone-water solution. The layered structure resulted from dissolution and recrystallization of CaHAP. This structure disappeared on outgassing above 200 °C accompanying the remove of alkyl groups and H2O and the formation of mesopores and new surface P-OH groups. Acknowledgment. We thank Mr. Masao Fukusumi of Osaka Municipal Technical Research Institute for his help with the TEM measurements. This study was supported in part by Nippon Sheet Glass Foundation for Materials Science and Technology and the Grants-in-Aid for Scientific Research (B and C) of the Ministry of Education, Science, Sports and Culture, Japan. LA960422F (18) Dollimore, D.; Heal, G. R. J. Appl. Chem. 1964, 14, 109. (19) Lippens, B. C.; de Boer, J. H. J. Catal. 1965, 4, 319.