Langmuir 1999, 15, 6557-6562
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Formation of Calcium Phosphates in Aqueous Solutions in the Presence of Carbonate Ions John Kapolos and Petros G. Koutsoukos* Institute of Chemical Engineering and High Temperature Chemical Processes (ICE/HT-FORTH) and Department of Chemical Engineering, University of Patras, GR 265 00 Patras, Greece Received September 18, 1998. In Final Form: May 3, 1999 The role of carbonate on the kinetics and physicochemical characteristics of the crystal growth of stoichiometric hydroxyapatite was investigated at 37 °C, 0.15 M NaCl ionic strength, and pH 7.40 at constant supersaturation. It was found that at low supersaturations the presence of carbonate ions at concentration as high as 0.1 mM had no effect on the crystal growth kinetics while at higher supersaturation (>3.8) the presence of carbonate ions resulted in reduction of the crystal growth rates and changed the dependence of the rates of crystal growth on the solution supersaturation from parabolic to linear. Carbonate was incorporated to the apatite growing on the seed crystals and caused changes in their morphology, favoring platelike formations.
Introduction The biologically important apatitic minerals encountered in the hard tissues of higher mammals and humans contain significant amounts of carbonate.1-3 The incorporation of carbonate in the apatitic lattice may result in structural deformations, and the presence of carbonate in the mineral phase may be identified by spectroscopic methods (X-ray diffraction and infrared spectroscopy).4-7 Since the formation of biological apatites takes place in biological fluids supersaturated with respect to calcium phosphate, the solution composition and especially the carbonate content are a determining factors for the kinetics of formation and the morphological and physicochemical characteristics of the mineral phase.8 The nature of the mineral phase formed by crystallization in supersaturated solutions is determined by both thermodynamics and kinetics. Thermodynamics calculations aim at estimating the driving force for the formation of the mineral phase and are done taking into account all the solution equilibria.9 Little work has been done concerning the kinetics of crystal growth of apatite in the presence of carbonate ions. Earlier work was done in carbonate-containing buffer solutions10-12 while more recently published results at one supersaturation and over a very limited partial pressure of carbon dioxide, to avoid significant variation in the solution’s supersaturation, showed a drastic reduction of the rates of hydroxyapatite (Ca5(PO4)3OH, HAP) (1) Young, R. A. J. Dent. Res. (Suppl.) 1974, 53, 193. (2) Simpson, D. R. Clin. Orthop. Relat. Res. 1972, 86, 260. (3) Young, R. A. Colloid Interface CNRS (Paris) 1973, 21. (4) LeGeros, R. Z.; LeGeros, J. P.; Trautz, O. R.; Shirra, W. P. Adv. X-ray Anal. 1971, 14, 57. (5) Suzuki, M. Colloid Interface CNRS (Paris) 1973, 77. (6) Elliot, J. C. The crystallographic structure of dental enamel and related apatites. Ph.D. Thesis, University of London, 1964. (7) Bonel, G.; Montel, G. C. R. Acad. Sci. [D] (Paris) 1964, 258, 923. (8) Iijima, M.; Kamemizu, H.; Wakamatsu, N.; Goto, T.; Doi, Y.; Moriwaki, Y. J. Cryst. Growth 1994, 135, 224. (9) Koutsoukos, P. G. In Calcium phosphates In Biological and Industrial Systems; Amjad, Z., Ed.; Kluwer Academic Publishers: Boston, 1998. (10) Simpson, D. R. Am. Mineral. 1967, 52. (11) Hayek, E.; Konetscny, H.; Schnell, E. Angew. Chem. 1966, 78. (12) Bachra, B. N.; Trautz, O. R.; Simon, S. L. Arch. Biochem. Biophys. 1963, 103, 124.
crystal growth up to 87% for PCO2 ) 10 Torr.13 In the present work we have attempted to investigate the kinetics of crystal growth of stoichiometric hydroxyapatite in the presence of carbonate ions, at conditions of constant supersaturation. We have used the closed system approach14 in order to avoid the slow equilibration process between the gas and the aqueous phase. It was thus possible to make quantitative estimates not only of the role of the carbonate ions on the kinetics of crystal growth but also of the interaction of the carbonate ions with the HAP solid phase. The experiments reported were done at constant supersaturation, which allows for the kinetics measurements to be done at well-defined steady-state conditions. Thus, in addition to the accurate measurements of the rates of crystal growth of HAP, the mineral composition with respect to calcium, phosphate, and carbonate may be deduced. The potential incorporation of carbonate ions in the HAP lattice would lead to equivalent deviations from the steady state maintained by the addition of titrants with the stoichiometry of HAP.15 Thus, the solution composition during the crystal growth of HAP was used as an indirect probe of the interaction of carbonate ions at the HAP/aqueous solution interface. Experimental Section All experiments were performed at 37.0 ( 0.1 °C in a thermostated, double walled, water-jacketed Pyrex glass reactor, and the reaction mixtures were stirred by a magnetic stirrer with a Teflon coated stirring bar. The volume of the reactor was 0.22 dm3. Crystalline solid, reagent grade, calcium chloride, sodium dihydrogen phosphate, sodium chloride, sodium hydrogen carbonate (Merck), and triply distilled, CO2-free water were used in the preparation of solutions. Sodium hydroxide solutions were prepared from a concentrated standard (Merck, Titrisol). The stock solutions of calcium were standardized with ion chromatography (Metrom IC 690) and atomic absorption spectroscopy (Perkin-Elmer 305 A). The sodium phosphate stock solutions were standardized by spectrophotometric analysis for total phosphate16 and by volumetric titrations with standard sodium hydroxide solutions using thymol blue indicator. (13) Campbell, A. A.; LoRe, M.; Nancollas, G. H. Colloids Surf. 1991, 54, 25. (14) Stum, W.; Morgan, J. Aquatic Chemistry: J. Wiley: New York, 1981. (15) Heughebaert, J. C.; Nancollas, G. H. J. Phys. Chem. 1984, 88, 2478.
10.1021/la981285k CCC: $18.00 © 1999 American Chemical Society Published on Web 06/30/1999
6558 Langmuir, Vol. 15, No. 19, 1999 The supersaturated solutions were prepared in the thermostated reactor by mixing equal volumes of calcium chloride and sodium dihydrogen phosphate. The ionic strength was adjusted to 0.15 mol‚dm-3 by the addition of the appropriate amount of standard sodium chloride solution. Immediately after the mixing of the solutions, the working solution was sealed by a perspex lid with holes accommodating two electrodes, a nitrogen inlet, a sampling port, and titrant delivery ports. The pH was adjusted by the slow addition of standard sodium hydroxide solution and the working solution was allowed to deaerate by bubbling nitrogen water vapor saturated in the absence of carbonate through a sparger. The pH was monitored with a pair of glass/saturated calomel electrodes standardized before and after each experiment with NBS standard buffer solutions of pH 6.864 and 7.414.17 The crystal growth process was initiated by the addition of known quantities of well-characterized HAP seed crystals prepared by a method described elsewhere.3,4 The specific surface area of the seed crystals, as determined by a multiple-point BET method, was found to be 29.0 m2 g-1. The stoichiometric calcium to phosphate molar ratio Ca/P was determined following dissolution of accurately weighted solid specimens in 0.1 N HCl and subsequent analysis for total calcium and total phosphate. For the HAP seed crystals it was found to be 1.65 ( 0.02. Since our experiments were done using NaCl as inert electrolyte, the solids obtained were analyzed for Na content by atomic absorption spectrometry, following dissolution in acid. Moreover, additional examination for the presence of Na in the solids was performed by XRF, EDX microanalysis (JEOL-LINK), and ion chromatography (Dionex DX120). After the addition of the HAP seed crystals in the supersaturated solutions, crystal growth started immediately without any induction time. The formation of the solid phase was accompanied with a pH drop. The solution supersaturation was kept constant by the addition of titrant solutions consisting of calcium chloride, sodium dihydrogen phosphate, sodium hydroxide, and sodium chloride with concentrations corresponding to the stoichiometry of HAP, that is, Ca/P/OH ) 5:3:1, where Ca, P, and OH denote the respective molar ratios.18 The two titrant solutions were placed in two mechanically coupled burets, and their addition was controlled by the hydrogen ion probe connected to a pH stat delivery unit (Radiometer). Provided that Cat and Pt were the total calcium and total phosphate conentrations in the working solution, respectively, CNaOH the final alkali concentration added to the supersaturated solutions for the pH adjustment, and CNaCl the salt concentration used for the adjustment of ionic strength, the composition of the titrant solutions was as follows:
buret 1: [mCat + 2Cat] M CaCl2
[5m3 Ca + 2P ] M NaH PO 5 1 b. ( mCa + 2 Ca + 2C ) M NaOH 5 3
buret 2: a.
t
NaOH
t
2
t
4
NaOH
c. 2[I - mCat] M NaCl The value of m in the experiments of the present work was taken as m ) 10 and was determined from preliminary experiments. The value of m depends on the rate of formation of the solid and is related to the supersaturation level and the total surface area of the inoculating seed crystals. The choice of m is such that no over- or undershooting of the set point is done during the crystal growth process with respect to the solution pH, thus achieving optimization of the constant-composition maintenance. The rates of crystal growth were measured from the profiles of the volumes of titrant solutions added as a function of time at the point corresponding to 1% of crystal growth with respect to the amount of the inoculating seed crystals. During the crystallization process, samples were withdrawn and filtered through membrane filters (Millipore 0.22 µm). The (16) Gee, A.; Deitz, V. R. Anal. Chem. 1953, 25, 1320. (17) Bates, R. G. pH Determination; J. Wiley: New York, 1973. (18) Koutsoukos, P. G.; Amjad, Z.; Tomson, M. B.; Nancollas, G. H. J. Am. Chem. Soc. 1980, 102, 1553.
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Figure 1. Kinetics of crystal growth of HAP on HAP seed crystals in the absence and in the presence of carbonate ions at constant supersaturation. Plot of the crystal growth rates as a function of the relative solution supersaturation with respect to HAP at 37 °C, pH 7.40, 0.15 M NaCl: (O) blank; (0) 5 µM; (4) 100 µM NaHCO3. (9) Apatite growth on HAP (4.94% carbonate) in the presence of 1.8 × 10-5 M carbonate (ref 13). filtrates were analyzed for calcium by ion chromatography and for phosphate spectrophotometrically to confirm the constancy of the solution composition. Moreover, at the end of each experiment the solution was filtered, and the solid was dried at room temperature and was examined by infrared spectroscopy (Perkin-Elmer 16PC FTIR), powder X-ray diffraction (Philips 1830/40), and scanning electron microscopy (JEOL JSM 5200). Chemical analysis was also done to determine the Ca/P molar ratio of the crystals grown in the supersaturated solutions. For the HAP crystal growth in the presence of CO32- the same experimental procedure was applied, except for nitrogen bubbling, which was omitted to avoid carbonate depletion. Moreover, at the concentration levels used here, according to our thermodynamic calculations, CO2 exchange even for an open system would be minimal. According to these calculations, the equilibrium pH at the experimental conditions is calculated including the amount of standard base needed to bring the pH to the desired value. The calculations are based on the thermodynamic stability constants of the equilibria involved in the mass balance equations for total calcium, phosphate, and carbonate and the electroneutrality condition. A small amount of NaHCO3 solution, prepared before each experiment, was added to the working solution. The final concentrations of NaHCO3 in the working solutions were between 5 × 10-6 and 1 × 10-4 M. In all experiments HAP was added as a slurry of stoichiometric HAP crystals suspended in a saturated solution (slurry density: 20.8 ( 0.2 mg/mL).
Results and Discussion Crystallization in calcium phosphate supersaturated solutions may result in the formation of a number of phases, in the following order of decreasing solubility: calcium phosphate dihydrate (CaHPO4‚2H2O, DCPD), octacalcium phosphate (Ca4H(PO4)3‚2.5H2O, OCP), and HAP. The concentrations at 37 °C are 1.87 × 10-7 M,2,19 10-49.3 M,8,19 and 2.35 × 10-59 M,9,2119-21 respectively. The driving force for the formation of any of the above crystalline solids is the difference of the chemical potential of the supersaturated solution, µs and that at equilibrium, µ∞, respectively. Provided that the standard chemical potentials of the two states are the same, the driving force per mole may be expressed as the respective change in the Gibbs free energy for the formation of a calcium phosphate phase with the stoichiometry (19) Marshall, R. Ph.D. Thesis, SUNY at Buffalo, 1970. (20) Shyu, L. J.; Perez, L.; Zawacki, S. J.; Heughebaert, J. C.; Nancollas, G. H. J. Dent. Res. 1981, 62, 398. (21) McDowell, H.; Gregory, T. M.; Brown, W. E. J. Res. Natl. Bur. Stand., Sect. A 1977, 81, 273.
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Table 1. Crystallization of HAP Seed Crystals in the Presence and in the Absence of Carbonate Ions at Conditions of Constant Supersaturation; pH 7.40, 37° C, 0.15 M NaCla Cat/ Pt/ [carbonate]/ ×10-4 ×10-4 ×10-5 exp M M M 1 11 12 13 2 21 22 23 24 3 31 32 33 4 41 5 51 52 53
2.0 2.0 2.0 2.0 3.0 3.0 3.0 3.0 3.0 4.0 4.0 4.0 4.0 4.5 4.5 5.0 5.0 5.0 5.0
1.2 1.2 1.2 1.2 1.8 1.8 1.8 1.8 1.8 2.40 2.40 2.40 2.40 2.7 2.7 3.0 3.0 3.0 3.0
0.5 5.0 10.0 0.5 5.0 7.0 10.0 0.5 5.0 10.0 0.5 0.5 5.0 10.0
σ
Rg/×10-9 crystallization wrt seed mol m-2 crystal min-1
1.53 1.53 1.53 1.53 2.63 2.63 2.63 2.63 2.62 3.67 3.67 3.67 3.67 4.16 4.16 4.67 4.67 4.67 4.66
1.9 6.8 5.0 3.4 7.3 10.0 8.0 6.2 3.8 10.0 14.2 14.4 14.6 23.2 16.1 38.5 16.1 14.8 15.8
4 11 9 5 10 12 11 8 8 15 17 14 15 30 28 36 22 16 20
Figure 3. Expansion of the FTIR spectra presented in Figure 3 between 1360 and 1540 cm-1. Table 2. Ca/P Molar Ratios Determined by Chemical Analysis of the Solid Precipitates in the Absence and in the Presence of Carbonate Ions at Constant Supersaturation
a Initial conditions: relative supersaturation with respect to HAP and measured rates of crystal growth.
exp
Ca/P molar ratio
1 11 2 21 4 41 5 51 52 53
1.67 ( 0.01 1.67 ( 0.02 1.67 ( 0.01 1.68 ( 0.02 1.65 ( 0.02 1.74 ( 0.01 1.67 ( 0.01 1.70 ( 0.01 1.72 ( 0.01 1.72 ( 0.02
The results of the seeded growth experiments done at various supersaturations and at different carbonate concentrations are summarized in Table 1. Plots of the rate of HAP crystal growth Rg versus the relative solution supersaturation according to the theory of crystal growth from solution may be described by equations of the type22
Rg ) kσn Figure 2. Infrared spectra of (a) HAP seed crystals (b) HAP grown on HAP in the presence of 5 × 10-5 M NaHCO3, and (c) HAP grown on HAP in the presence of 1 × 10-4 M NaHCO3.
Caν1(PO4)ν2OHν3Hν4 with ν1 + ν2 + ν3 + ν4 ) ν and is computed from eq 1: ν1 ν2 ν3 ν4 RT aCa2+aPO43-aOH-aH+ ln ∆G ) ν K°s
(1)
where R is the gas constant, T is the absolute temperature, ai are the activities of the subscripted ions, and K°s is the thermodynamic solubility product of the mineral for which the change in Gibbs free energy is computed. The ratio in the logarithmic term of eq 1 is defined as the supersaturation ratio Ω:
Ω)
ν3 ν4 ν1 ν2 aCa 2+aPO 3-aOHaH 4
K°s
(2)
The relative supersaturation σ is
σ ) Ω1/ν - 1 For HAP ν1 ) 5, ν2 ) 3, ν3 ) 1, and ν4 ) 0 (ν ) 9).
(3)
(4)
Kinetics plots of the rates of crystal growth of HAP in the absence and in the presence of carbonate ions according to eq 4 are shown in Figure 1. As may be seen, in the absence of carbonate ions, the dependence of the crystal growth rates on supersaturation was parabolic, while in their presence it was linear. According to the BurtonCabera-Frank (BCF) model, a parabolic dependence and a linear dependence of the rates of crystal growth are predicted for crystal growth at low and high supersaturations, respectively.23 As may be seen from the kinetics plots shown in Figure 1, the presence of carbonate ions in the supersaturated solutions over a wide range of concentrations (5 µM to 0.1 mM), although it affected very little the relative magnitude of the rates of HAP crystal growth, resulted in the change of the mode of dependence of the rate on the relative supersaturation with respect to HAP from parabolic to linear. Since no mechanism change is anticipated (the values of the rates remained of the same order of magnitude), it was assumed that the change of the apparent reaction order from n ) 2 to n ) 1 could be due to changes in the surface composition and structure of the solid substrate, providing the active sites (22) Garside, J.; So¨hnel, O. Precipitation; Butterworth-Heinemann: London, 1993. (23) Nancollas, G. H. In Biomineralization; Mann, S., Webb, J., Williams, R. T. P., Eds.; VCH: Weinheim, 1989; pp 157-187.
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Figure 4. Powder X-ray diffraction spectra of (a) HAP (stoichiometric) grown on HAP seed crystals and carbonated apatite grown on HAP seed crystals in the presence of (b) 50 µM or (c) 100 µM.
for crystal growth. It should be noted that the rates of crystal growth of HAP in the presence of carbonate were measured from the traces of titrants added as a function of time at the point corresponding to extents of crystal growth sufficiently low to preclude any significant changes in the solid solubility due to the incoporation of carbonate ions. In Figure 1 it should also be noted that the measured rates of crystal growth were in agreement with values reported in the literature for the HAP growth on carbonated apatite in the presence of 1.8 × 10-5 M carbonate (partial pressure of CO2: 10.18 Torr).13 As may be seen from the kinetics plot shown in Figure 1, two regions are defined in the domain of the dependence of the rates of crystal growth on the solution supersaturation: one in which the presence of carbonate had an accelerating effect (low supersaturation region) and a second in which the presence of carbonate ions had an inhibitory function. Raising the carbonate concentration above 0.1 mM was avoided in order to keep solutions undersaturated with respect to the calcium carbonate polymorphs. Moreover, all solutions employed were supersaturated only with respect to HAP in order to preclude the formation of other calcium phosphate precursor phases which could possibly complicate interpretation of the kinetics results. All experiments reported in this work proceeded to mass deposition with respect to the amount of the inoculating seed crystals between 10 and 30%, as may be seen in Table 1. The deviations of calcium and phosphate concentrations from the values corresponding to constant solution composition were within the experimental error, not allowing for a reliable quantitative estimate of the possible carbonate incorporation in the solid formed, on the basis of the solution composition. The spectroscopic examination of the solids formed in the presence of carbonate showed the characteristic carbonate bands at 1500, 879, and 716 cm-1, from which a maximum incorporation of about 5% in the solid was estimated.24 Typical infrared spectra of carbonated apatites obtained by crystallization of HAP in the presence of carbonate at conditions of constant supersaturation are shown in Figure 2. Unfortunately, due to the very low extent of carbonate incorporation, the carbonate bands cannot be clearly distingushed, especially
when the full spectrum is presented. The expansion of the spectrum between 1400 and 1500 cm-1 shows the carbonate bands of the apatites grown in the presence of carbonate ions.24 These bands were used to obtain the approximate quantitative estimate of carbonate incorporation. The weak bands around 1200 cm-1 are possibly either due to in-plane bending modes of interphosphate or due to water to orthophosphate hydrogen bonds.25,26 The presence of the HOPO3 stretching mode is also possible27 but less likely in view of the stoichiometry of the apatite seed crystals.28 It is reported that the bands at 1430 and 1455 cm-1 (both of which may be seen in Figure 3b and c) are due to CO32- ions substituting for PO43-.29 Moreover, assignment of the weak bands around 1200 cm-1 to pyrophosphate is very unlikely, since the specimens (both carbonated and seed crystals) were not heated. Drying at room temperature does not induce the formation of pyrophosphate.27 We have further proceeded with the chemical analysis of the solid phases collected at the end of the crystal growth experiments in the absence and in the presence of the carbonate ions. The results are summarized in Table 2. As may be seen, in the presence of higher concentrations of carbonate, there was a tendency of increasing Ca/P molar ratio. This trend may be ascribed to reduced values of phosphate content due to their substitution by carbonate. It should be noted that chemical analysis under our conditions did not show the presence of Na in the grown HAP. The progressive substitution may be seen in the powder XRD spectra shown in Figure 4. The evidence of carbonate incorporation in the apatite lattice is clearly seen in the spectrum of the precipitate formed in the presence of 0.1 mM NaHCO3. The effect of (24) Nelson, D. G. A.; Featherstone, J. D. B. Calcif. Tissue Int. 1982, 34, 569. (25) Ryski, Y. I.; Stavitskaya, G. P. Opt. Spectrosc. (USSR) Engl. Transl. 1960, 8, 320. (26) Chapman, A. C.; Thirlwell, L. E. Spectrochim. Acta 1964, 20, 937. (27) Fowler, B. O.; Moreno, E. C.; Brown, W. E. Arch. Oral Biol. 1966, 11, 477. (28) Arends, J.; Davidson, C. L. Calcif. Tissue Res. 1975, 18, 65. (29) Arends, J.; Jongebl’oed, W. L. Recl. Trav. Chim. Pays-Bas 1981, 100, 3.
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Figure 5. Scanning electron micrographs of (a) HAP seed crystals, (b) HAP grown on HAP seed crystals at constant supersaturation, (c and d) carbonated apatite grown on HAP seed crystals in the presence of 5 × 10-5 M NaHCO3 total calcium, 0.1 mM; total phosphate, 0.06 mM; pH 7.40, 0.15 M NaCl, 37 °C; (e and f) carbonated apatite grown on HAP seed crystals in the presence of 0.1 mM NaHCO3 + 0.15 M NaCl (pH 7.40; 37 °C; total calcium, 0.3 mM; total phosphate, 0.18 mM).
carbonate is reported to affect the size and shape8,30-32 of the grown carbonated apatite crystals. The presence of carbonate in the aqueous supersaturated solutions resulted in the formation of platelike crystallites coexisting with needle-like crystallites as the seed crystals, as may be seen in the scanning electron micrographs shown in Figure 5. HAP seed crystals consisted of needle-like (30) Legeros, R. Z.; Trantz, O. R.; Legeros, J. P.; Klein E. Bull. Soc. Chim. 1968, 1712. (31) Barralet, J. E.; Best, S. M.; Bonfield, W. Bioceramics 1993, 6, 179. (32) Bachra, B. N. Ann. N. Y. Acad. Sci. 1963, 109, 251.
crystallites, mean size 0.7 µm, which upon crystallization at conditions of constant supersaturation retained their morphology, growing in the direction of the c axis (Figure 5b). The presence of carbonate ions caused changes in the morphology of the crystals which showed a platelike habit. Changes of the HAP crystal morphology in the presence of carbonate have been reported in the literature,8,30,31 and they are possibly related with the incorporation of the ions in the crystals grown from supersaturated solutions in their presence. Besides habit changes of the HAP crystals, the presence of carbonate may also have an
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effect on the mineral solubility.27 From the kinetics of HAP crystallization, it may also be suggested that carbonate incorporation is related to the relative solution supersaturation. At higher supersaturations it is possible that the change of the apparent order from 2 to 1 may be due to the formation of carbonated apatites on the surface of the growing HAP seed crystals.33,34 Conclusions The effect of the presence of carbonate ions in supersaturated calcium phosphate solutions in which the crystal growth of HAP seed crystals was investigated at pH 7.40, (33) Shirkhanzadeh, M. Ann. N. Y. Acad. Sci. 1963, 109, 251. (34) Cheng, Z. H.; Yasukawa, A.; Kandori, K.; Ishikawa, T. J. Chem. Soc., Faraday Trans. 1998, 94, 1501.
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37 °C, and 0.15 M NaCl showed that these ions caused a reduction of the rates of HAP crystal growth past a supersaturation threshold. Carbonate ions were incorporated into the HAP lattice, substituting phosphate ions, and resulted in changes in the morphology of the HAP crystallites which showed a platelike habit. The incorporation of carbonate in the apatite lattice was further evidenced from the increase of the molar Ca/P ratio from 1.67 to 1.74. Acknowledgment. Partial support of this work by the General Secretariat of Research and Technology (GSRT) through a PENED program is gratefuly acknowledged. LA981285K
