Energetics of Calcium Phosphate Nanoparticle Formation by the

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J. Phys. Chem. C 2009, 113, 5478–5484

Energetics of Calcium Phosphate Nanoparticle Formation by the Reaction of Ca(NO3)2 with (NH4)2HPO4 Miroslav Leskiv,† Ana L. C. Lagoa,† Henning Urch,‡ Janine Schwiertz,‡ Manuel E. Minas da Piedade,*,† and Matthias Epple‡ Departamento de Quı´mica e Bioquı´mica, Faculdade de Cieˆncias, UniVersidade de Lisboa, 1649-016 Lisboa, Portugal, and Inorganic Chemistry and Center for Nanointegration Duisburg-Essen (CeNIDE), UniVersity of Duisburg-Essen, UniVersita¨tsstrasse 5-7, 45117 Essen, Germany ReceiVed: January 14, 2009; ReVised Manuscript ReceiVed: February 17, 2009

The energetics of the reaction between Ca(NO3)2(aq) and (NH4)2HPO4(aq) leading to the formation of calcium phosphate nanoparticles was investigated by flow calorimetry. The relationship between the observed enthalpy change, the pH of the (NH4)2HPO4(aq) solution, and the elemental composition and morphology of the obtained compounds was studied. Results of elemental analysis, combined thermogravimetry-infrared spectroscopy, X-ray powder diffraction, and scanning electron microscopy showed that the change of the pH during the precipitation reaction through the addition of controlled amounts of NaOH(s) to the (NH4)2HPO4(aq) solution leads to significant and reproducible changes of the chemical composition, morphology, and amorphous character of the obtained materials. These changes are reflected by the corresponding enthalpy of reaction, which seems to be predominantly determined by the differences in chemical composition. Introduction Calcium phosphates are the inorganic component of human hard tissue, e.g., of bone, teeth, and tendons.1-7 Except in the case of enamel, biological calcium phosphates always occur as nanocrystals of the so-called “biological apatite”, which consists of a carbonated hydroxyapatite with the approximate formula Ca10-x(PO4,CO3)6-x(HPO4)x(OH)2-x.8-14 The formation of bone occurs in a continuous process called “remodeling” that involves a permanent dissolution and reprecipitation of calcium phosphate. Osteoblasts are the cells which are involved in bone formation,15 and osteoclasts are responsible for bone resorption.16,17 The latter process is accomplished by forming an acidic compartment between the cell and the bone, wherein the calcium phosphate is dissolved.17 Although calcium phosphate bioceramics of high crystallinity, such as calcined stoichiometric hydroxyapatite, Ca10(PO4)6(OH)2, show a good biocompatibility, the reported biodegradation rate is much lower than required by the bone remodeling process.18,19 Thus when this type of material is used for bone repair, the implant remains unchanged at the implantation site for years after the operation, a situation that is not desirable from a clinical point of view. It has been suggested that inorganic materials that most closely mimic the nanosized bone structure and surface are best suited for bone replacement/repair.20-22 With this in mind, we have previously studied the morphology and structure of biomimetic calcium phosphate nanoparticles to be used in bone implants, which were prepared by rapid precipitation from aqueous solution.22,23 This type of particle has also found other interesting applications. For example, when functionalized with polymers they can be used for surface coating of implant materials, thus improving their biocompatibility.24,25 If they are functionalized by nucleic acids (DNA or RNA), they can act as transfection agents, i.e., they are taken up by living cells * To whom correspondence should be addressed. E-mail: [email protected]. † Universidade de Lisboa. ‡ University of Duisburg-Essen.

and the nucleic acid influences the intracellular protein synthesis.26-31 Despite these important potential applications, systematic investigations of the energetics of reactions leading to the formation of unfunctionalized or functionalized calcium phosphate nanoparticles are lacking. These studies are, however, very helpful to define strategies for a controlled production of materials tailored for specific applications. Here, we report a calorimetric investigation of the reaction between Ca(NO3)2(aq) and (NH4)2HPO4(aq), which has been widely used in the synthesis of calcium phosphate nanoparticles.23,32,33 The study was focused on the relation between the observed enthalpy change, the pH of the reactant solutions, and the elemental composition and morphology of the obtained calcium phosphate. Materials and Methods General. Elemental analyses (C, H, N) were made on a CE instruments EA1110 apparatus. The Ca and Na contents of the nanoparticle samples were determined by atomic absorption spectroscopy after acidic dissolution using a Thermo Electron Corporation M-Series instrument. The corresponding amounts of phosphate were obtained by the method of Gee and Deitz34 using a Varian Cary WinUV spectrophotometer. X-ray powder diffraction experiments were performed with a STOE transmission diffractometer STADI P 2003-10 with Cu KR radiation (λ ) 1.54 Å) in glass capillaries. The pH measurements were made with a resolution of ( 0.001 using a Radiometer TIM 900 apparatus and a Radiometer Red Rod pH electrode. Combined thermogravimetry-infrared analyses (TG-IR) were carried out on a Netzsch STA 409/Bruker Vertex 70 system. The experiments were performed from 30 to 1000 °C, with a heating rate of 3 K min-1, under dynamic oxygen atmosphere (50 mL min-1) and in open Al2O3 crucibles. The sample mass was ∼30 mg. Scanning electron microscopy (SEM) was performed with a FEI ESEM Quanta 400 FEG instrument on Au/Pd-sputtered samples. Materials. The solutions used in the experiments were freshly prepared from distilled water and Ca(NO3)2 · 4H2O (Riedel-de

10.1021/jp900399c CCC: $40.75  2009 American Chemical Society Published on Web 03/18/2009

Energetics of Calcium Phosphate Nanoparticle Formation

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TABLE 1: Characterization of the Nanoparticles Obtained from the Reaction Ca(NO3)2 + (NH4)2HPO4 by Elemental Analysis and Thermogravimetric (TG) Analysisa elemental analysis [NaOH]/mM 0 4.1 7.9 12.1 20.7 32.2 45.7

TG

w(Ca)/%

w(PO34 )/%

w(H)/%

w(Na)/%

w(CO2)/%

w(H2O)/%

34.25 34.69 34.87 36.67 34.60 33.14 33.98

55.93 56.20 56.05 54.78 51.75 49.70 48.32

0.87 0.98 0.98 1.00 1.52 1.77 1.80

0 0 0 0 0 0.19 0.22

0.64 0.71 1.11 1.01 1.02 1.25 1.31

1.33 5.61 7.40 8.92 12.05 15.02 14.70

a The symbol w refers to mass percent, and [NaOH] is the molar concentration of NaOH in the initial ammonium hydrogen phosphate solution.

Ha¨en, g99%), (NH4)2HPO4 (May & Baker, g97%), and NaOH (Pronalab, p.a.). The use of distilled water or of distilled and deionized water from a Millipore system (conductivity 0.1 µS) in the preparation of the solutions had no detectable effect on the calorimetric results. Flow Calorimetry. Calorimetric measurements were carried out at 298.15 K with a modified version of an LKB 10700-1 flow microcalorimeter.35,36 A typical experiment involved the recording of two measuring curves corresponding to the electrical calibration and the reaction, respectively. Each measuring curve consisted of a plot of the differential output of the thermopiles versus time. In the calibration, a constant potential of V volts, set in the power supply, was applied to the resistance inside the measuring cell, causing a current of intensity I amperes to flow for t seconds. The heat, Q, dissipated by Joule effect inside the cell during the main period of the calibration was calculated from:

Q)

∑ ViIi∆ti

(1)

i

where Vi and Ii are the ith voltage and current readings by the power supply and the digital multimeter, respectively, and ∆ti is the time difference between two consecutive data acquisitions. The calibration constant ε was obtained from:

ε)

Q Ac

(2)

where Ac is the area of the corresponding measuring curve. The standard molar enthalpy of the reaction under study, ∆rHom, was derived through the equation: o ∆rHm )ε

As n

(3)

where ε is the mean value of the calibration constant, As is the area of the corresponding curve, and n is the amount of substance in moles of the reference compound that reacted during the main period. The value of n was obtained from the experimentally determined mass flow. It should finally be noted that tests carried out for some of the experiments showed that basing the calculations on the area of the measuring curves eq 2 and eq 3, instead of the conventional displacements of the signals from the baseline,35,36 had no effect on the results. Reaction of Ca(NO3)2 with (NH4)2HPO4. The reaction of calcium nitrate with ammonium hydrogen phosphate leading

to the calcium phosphate nanoparticles was first studied outside the calorimeter under conditions close to those present in the calorimetric experiments. In this case, the synthesis of the nanoparticles was performed by mixing 100 mL of 0.018 M Ca(NO3)2(aq) with 100 mL of 0.0108 M (NH4)2HPO4(aq), which corresponds to a reaction mixture with the same Ca/P molar ratio of stoichiometric hydroxyapatite (Ca/P ) 1.67). The pH of the (NH4)2HPO4 solution was changed in different experiments by the addition of known amounts of solid NaOH. The reaction mixture was stirred with a glass rod for ∼2 min, and the precipitated nanoparticles were filtered in vacuum through a membrane filter (Pall Life Sciences Supor R-100; pore size 0.1 µm). The filtration lasted for an additional ∼10 min. High and low limits of the mass of product formed were determined as follows: The “high limit mass”, mhl, was calculated from the difference between the initial mass of the filter and the mass of the filter with the retained product after drying for 1-2 days in air at room temperature. This mass is an upper limit of the “true” mass of nanoparticles obtained in the reaction because complete removal of the water content could not be achieved. The “low limit mass”, mll, was taken as the mass of solid collected from the filter and subsequently dried in vacuum for about two days at ∼298 K. Because it was impossible to quantitatively collect all compound retained in the filter, mll represents a lower limit of the mass of nanoparticles formed. The vaccuum-dried material was analyzed by elemental analysis and thermogravimetry (see Results and Discussion). In the calorimetric experiments the calorimeter was first allowed to stabilize while a freshly prepared 0.018 M aqueous solution of Ca(NO3)2 of known pH and distilled water were pumped into the mixing cell at a flow of approximately 20 mL · h-1 each. Data collection was started after this stabilization period, and a well-defined baseline was obtained while maintaining the water and calcium nitrate solution flowing into the mixing cell. The reaction was initiated by replacing the water flow by a flow of a freshly prepared 0.0108 M aqueous solution of (NH4)2HPO4, which was maintained during a known period of time. As in the case of the preliminary studies mentioned above, the pH of the (NH4)2HPO4 solution was previously adjusted in the different runs by judicious additions of solid NaOH (thereby keeping the volume of the solution practically constant) and experimentally determined. The reaction was stopped by replacing the ammonium hydrogen phosphate solution by distilled water, and the signal was allowed to return to the baseline. The aqueous suspension of nanoparticles exiting the mixing cell was collected throughout the reaction, and its pH was measured. The amount of (NH4)2HPO4 solution that flowed through the mixing cell was determined from the mass loss of the corresponding storage flask. Changing the flow rate

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Leskiv et al.

Figure 1. TG curves of nanoparticle samples synthesized using (NH4)2HPO4 solutions with different NaOH concentrations (given in parentheses).

to ca. 10 mL · h-1 had no significant influence on the observed enthalpy of reaction. After each experiment a 0.1 M aqueous HCl solution was flowed through the apparatus to remove residues of the nanoparticle suspension. The heat flow was monitored during this exothermic process to ensure a full removal of the nanoparticles. After the signal returned to the baseline, the acid solution was replaced by distilled water to complete the cleaning of the apparatus. In the previous experiments the baseline was defined relative to the dilution of a Ca(NO3)2 solution. Because the physical properties of the calorimetric liquids (e.g., viscosity) may have an impact on the baseline, a more correct method would, in principle, consist of obtaining the baseline from a run where the nanoparticle suspension resulting from the reaction was recirculated through the two flow tubes going to the calorimetric cell.36-39 However, we observed considerable baseline fluctuations when this was attempted. Thus, to evaluate the effect of the baseline measurement on the observed enthalpy of reaction trends, experiments were also carried out where the initial baseline was defined by pumping 0.0108 M (NH4)2HPO4(aq) and distilled water through the mixing cell at a flow of approximately 20 mL · h-1. Results and Discussion The 2005 IUPAC-recommended standard atomic masses were used in the calculation of all molar quantities.40 The results of the elemental and TG-IR analysis of the vaccuum-dried nanoparticles obtained in the study of the Ca(NO3)2 + (NH4)2HPO4 reaction that preceded the calorimetric experiments are given in Table 1. The TG-IR measuring curves corresponding to materials produced using different NaOH concentrations in the (NH4)2HPO4 reactant solution are illustrated in Figure 1. The FT-IR analysis of the gases evolved from these samples during the heating ramp indicated that the first mass loss step in the approximate range 50-450 °C was due to the loss of water. Because the different water loss steps could not be separated, this includes surface bound water,41 lattice water,41 and water resulting from the condensation of HPO42- to diphosphate according to the reaction:41,42 42HPO24 (s) f P2O7 (s) + H2O(g)

(4)

The second step in the temperature range from 450 to 750 °C corresponds to CO32- decomposition to CO2.41,43 The mass percentages of H2O and CO2 in Table 1 are plotted against [NaOH] in Figure 2. Figures 1 and 2 show that the water loss

Figure 2. Mass percentages of water and carbon dioxide obtained in the TG analysis (see Table 1) plotted against the concentration of sodium hydroxide in the (NH4)2HPO4 solution, [NaOH].

Figure 3. SEM micrographs of the nanoparticle samples obtained by the reaction of Ca(NO3)2 and (NH4)2HPO4 using different concentrations of NaOH in the initial (NH4)2HPO4 solution: (a) [NaOH] ) 0 mM; (b) [NaOH] ) 12.1 mM; (c) [NaOH] ) 32.2 mM; (d) [NaOH] ) 45.7 mM.

becomes more important and eventually reaches a constant value as [NaOH] increases. This trend should be dominated by a raise in the amount of surface-bound and lattice water because the corresponding content in HPO42 - must decrease as the reactant (NH4)2HPO4 solution becomes more basic. The observation that the carbonate content increases with increasing basicity can be ascribed to a preferential uptake of CO2 from air at higher pH. For the samples which were precipitated at higher pH, the temperature of decarboxylation shifts to lower values (from 710 °C to about 450 °C). This is probably due to the decreasing crystallinity at higher pH (see below) which facilitates the release of carbon dioxide. The SEM micrographs in Figure 3 show that from a morphological point of view the sample precipitated without addition of sodium hydroxide to the [(NH4)2HPO4] solution differs from all other samples. The former is composed by a mixture of rod-like and approximately spherical particles (Figure 3a) whereas the latter (Figure 3b-d) consist of spherical particles only. The dimensions of the rod-like particles are ca. 200 × 50 nm and all spherical particles have diameters of ca. 50-80 nm. X-ray powder diffraction indicated that the sample with the rod-like particles was nanocrystalline whereas the remaining samples were essentially X-ray amorphous: For [NaOH] ) 0 mM, 4.1 mM, and 7.9 mM, the samples were all poorly crystalline; those corresponding to [NaOH] ) 12.1 mM and

Energetics of Calcium Phosphate Nanoparticle Formation

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Figure 5. Ca/P molar ratio in the of nanoparticles obtained in the reaction of Ca(NO3)2 with (NH4)2HPO4 as a function of the sodium hydroxide concentration in the ammonium hydrogen phosphate solution. Figure 4. X-ray powder diffractograms of the nanoparticle samples obtained from the reaction of Ca(NO3)2 with (NH4)2HPO4 using different concentrations of NaOH in the initial (NH4)2HPO4 solution: [NaOH] ) 0 mM, [NaOH] ) 12.1 mM, [NaOH] ) 32.2 mM. The peaks at the bottom are those calculated for hydroxyapatite (ICDD 84-1998).

20.7 mM were almost amorphous, and when [NaOH] ) 32.2 mM and 45.7 mM, completely X-ray amorphous materials were obtained. This is illustrated in Figure 4 for [NaOH] ) 0 mM, 12.1 mM, and 32.2 mM. The total masses of the obtained nanoparticle samples expressed as high, mhl, and low, mll, limit masses (see Materials and Methods), are given in Table 2 as a function of [NaOH] and the pH of the reactant (NH4)2HPO4 solution, pHP. Also included in Table 2 are the corresponding Ca/P molar ratios, derived from Table 1, and the results of their qualitative characterization in terms of crystallinity (from X-ray diffraction) and morphology (from SEM). The results in Table 2 and the plot in Figure 5 show that as [NaOH] increases the Ca/P ratio tends to 1.67 which matches the initial value present in all reaction mixtures and also in stoichiometric hydroxyapatite, Ca10(PO4)6(OH)2. This is consistent with the fact that the equilibrium 3HPO24 (aq) + OH (aq) h PO4 (aq) + H2O(1)

(5)

is progressively shifted to the right as the pH of the (NH4)2HPO4 solution is raised, thus increasing the amount of PO43- directly available for reaction with Ca(NO3)2. The pH increase also leads to an increase of the total mass of nanoparticles produced (larger mhl and mll). Hence, the reaction yield increases as the pH becomes more basic. This is illustrated in Figure 6 where the mass fractions of Ca and P reacted, defined as:

w(Ca))

m(Ca)f m(Ca)i

(6)

w(P))

m(P)f m(P)i

(7)

and given as percentages, are plotted against the concentration of NaOH in the (NH4)2HPO4 solution. In eqs 6 and 7, m(Ca)i and m(P)i represent the masses of calcium and phosphorus in the reaction mixture calculated from the masses of solid Ca(NO3)2 and (NH4)2HPO4 used in the preparation of the corresponding solutions; m(Ca)f and m(P)f are the masses of

Figure 6. Mass fractions of reacted calcium, w(Ca), and phosphorus w(P) for the Ca(NO3)2 + (NH4)2HPO4 system, as a function of the concentration of sodium hydroxide in the initial (NH4)2HPO4 solution, [NaOH]. (b) w(P) based on mhl; (2) w(Ca) based on mhl; (O) w(P) based on mll; (4) w(Ca) based on mll (see text).

calcium and phosphorus in the obtained products calculated from mll and mhl (Table 2) and the results of elemental analysis in Table 1. Figure 6 shows that w(P) is always higher than w(Ca) unless the Ca/P molar ratio of stoichiometric hydroxyapatite (1.67) is reached at high pH. This reflects the fact that an initial Ca/P ratio of 1.67 was set for all reaction mixtures. Therefore, (NH4)2HPO4 will act as the limiting reagent and only when a solid with an approximate hydroxyapatite stoichiometry is formed and the “excess” Ca present in solution is consumed. It is also apparent from Figure 6 that w(P) and w(Ca) become approximately constant above [NaOH] ∼ 12.1 mM. Thus, above this NaOH concentration, a limiting amount of produced solid is approximately reached, although the elemental composition of the obtained material still somewhat changes. The results of the calorimetric studies are summarized in Table 3, where [NaOH] represents the molar concentration of sodium hydroxide in the reactant (NH4)2HPO4 solution, pHP and pHCa are the experimentally determined pH values of the (NH4)2HPO4 and Ca(NO3)2 solutions, respectively, and pHf refers to the pH of the final nanoparticle suspension exiting the o values were calculated from eq 3 and calorimeter. The ∆rHm refer to the amount of substance of reacted phosphorus, n(P). The indicated uncertainties correspond to the mean deviation of at least two experiments. The table includes results from experiments where the baseline was obtained by passing Ca(NO3)2 and H2O through the calorimetric cell and also from runs where (NH4)2HPO4 replaced Ca(NO3)2 during the baseline acquisition. The values of n(P) were computed as n(P) ) ni(P)w(P), where ni(P) is the amount of substance of (NH4)2HPO4 that passed through the calorimetric cell during an experiment and w(P) is defined in eq 7. Two values of n(P)

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TABLE 2: High, mhl, and Low, mll, Limit Masses, Ca/P Molar Ratio, Crystallinity, and Morphology of the Nanoparticles Obtained from the Reaction of Ca(NO3)2 with (NH4)2HPO4 [NaOH]/mM 0 4.1 7.9 12.1 20.7 32.2 45.7 a

pHp

mll/mg

mhl/mg

Ca/P ratio

crystallinitya

particle morphologyb

8.047 8.844 9.176 9.520 10.415 11.593 11.962

56.03 85.41 111.90 149.85 160.78 162.92 175.51

74.53 117.44 141.63 190.51 201.99 201.50 209.66

1.46 1.46 1.47 1.53 1.59 1.62 1.67

poorly crystalline poorly crystalline poorly crystalline almost amorphous almost amorphous amorphous amorphous

rod-like and spherical spherical spherical spherical spherical spherical spherical

From X-ray diffraction (see text). b From SEM (see text).

TABLE 3: Enthalpies of the Reaction Ca(NO3)2 + (NH4)2HPO4a [NaOH]/mM

pHP

pHCa

pHf

Baseline Relative to the Dilution of Ca(NO3)2 0.0 8.017 5.655 6.177 1.2 8.305 5.717 2.0 8.513 5.734 6.361 2.4 8.563 5.642 3.9 8.766 5.672 4.2 8.844 5.826 6.549 5.8 8.953 5.694 6.0 9.044 5.818 6.920 6.8 9.040 5.767 7.379 7.8 9.208 5.627 7.640 10.0 9.319 5.609 7.983 11.5 9.409 5.700 8.218 13.6 9.558 5.799 8.522 15.6 9.750 5.813 8.768 17.4 9.945 5.787 8.934 19.3 10.136 5.871 9.074 24.8 11.043 5.561 9.405 33.2 11.742 5.656 10.833 38.0 5.610 11.070 44.7 5.646 11.485 Baseline Relative to the Dilution of (NH4)2HPO4 0.0 7.903 6.084 5.958 6.3 8.835 5.962 6.562 7.3 8.755 6.029 6.576 22.4 10.464 5.876 9.117 45.1 11.477

n(P)hl/µmol

o ∆rHm (hl)/ kJ · (mol of P)-1

n(P)ll/µmol

o ∆rHm (ll)/ kJ · (mol of P)-1

41.7 57.2 36.0 65.7 73.5 40.6 83.8 49.6 87.5 98.8 105.1 110.1 127.3 125.4 132.7 135.9 120.5 140.8 110.8 121.4

26.18 ( 0.60 20.00 ( 0.40 16.54 ( 0.33 13.99 ( 0.18 8.28 ( 0.14 7.83 ( 0.30 3.01 ( 0.40 2.68 ( 0.47 0.30 ( 0.10 -3.21 ( 0.05 -4.46 ( 0.14 -5.00 ( 0.24 -5.53 ( 0.11 -5.78 ( 0.16 -5.96 ( 0.14 -6.05 ( 0.17 -4.25 ( 0.05 3.22 ( 0.02 5.75 ( 0.04 10.41 ( 0.02

29.3 42.2 27.1 50.0 57.3 31.7 66.6 39.5 70.0 79.4 85.2 89.6 103.9 102.5 108.5 111.0 98.2 114.7 91.2 103.3

37.20 ( 0.80 27.08 ( 0.54 21.95 ( 0.44 18.40 ( 0.24 10.63 ( 0.18 10.01 ( 0.38 3.78 ( 0.49 3.37 ( 0.60 0.37 ( 0.12 -3.99 ( 0.06 -5.49 ( 0.17 -6.15 ( 0.30 -6.77 ( 0.14 -7.07 ( 0.20 -7.29 ( 0.17 -7.40 ( 0.21 -5.22 ( 0.06 3.95 ( 0.02 6.98 ( 0.05 12.22 ( 0.02

26.3 60.2 56.2 75.6 77.9

26.39 ( 0.13 1.09 ( 0.00 -0.72 ( 0.10 -6.26 ( 0.16 3.50 ( 0.07

37.3 75.5 70.1 92.7 91.3

37.50 ( 0.18 1.37 ( 0.00 -0.90 ( 0.12 -7.67 ( 0.19 4.10 ( 0.08

a [NaOH] represents the molar concentration of sodium hydroxide in the reactant (NH4)2HPO4 solution; pHP and pHCa are the experimentally determined pHs of the (NH4)2HPO4 and Ca(NO3)2 solutions, respectively, and pHf refers to the pH of the final nanoparticle suspension exiting the calorimeter; The term n(P) corresponds to the amount of substance of reacted phosphorus and the designations h1 and 11 refer to values computed from the high and the low limit masses of sample collected in the experiments carried out outside the calorimeter.

o and therefore of ∆rHm can be obtained for each experiment: o One, ∆rHm(hl), based on w(P)hl, and the other, ∆rHmo(ll), based on w(P)ll, which correspond to the high and low limit masses of sample collected in the experiments carried out outside the calorimeter. These were computed from:

w(P)hl ) 0.4017 + 66.666[NaOH] - 2403.3[NaOH]2+ 26457[NaOH]3

(8)

w(P)ll ) 0.2827 + 61.180[NaOH] - 2274.6[NaOH]2 + 26083[NaOH]3

(9)

where [NaOH] is the molar concentration of NaOH in the reactant (NH4)2HPO4 solution (see Table 3). Eqations 8 and 9 were obtained from fits to graphical representations of w(P)hl or w(P)ll against [NaOH] and reproduce the experimental data with a maximum deviation of 4%, and average deviations of 1.4% and 2.6%, respectively. It should also be noted that the

calculations of ∆rHom are based on the assumption that the extent of the reaction of Ca(NO3)2 with (NH4)2HPO4 is the same in the calorimetric experiments and in the preliminary studies carried out outside the calorimeter. o trends in Table 3 are illustrated in Figure 7 where The ∆rHm o o (hl) and ∆rHm (ll) pair of values are the averages of each ∆rHm plotted as a function of [NaOH]. The displayed error bars o o (hl) - ∆rHm (ll)]/ represent mean deviations computed as [∆rHm 2. Two major features are apparent in the curves of Figure 7: (i) They show minima at [NaOH] ∼ 20 mM (pHP ∼ 10), with the reaction being first endothermic and then exothermic and again endothermic as [NaOH] increases. (ii) For [NaOH] larger than ∼20 mM the solid line, corresponding to the Ca(NO3)2 + H2O baseline, increasingly diverges toward more positive ∆rHom values from the dashed line which refers to the [(NH4)2HPO4 + NaOH] + H2O baseline. The divergence between the two curves that occurs above [NaOH] ∼ 20 mM is essentially due to a baseline effect. When the baseline is defined relative to the [(NH4)2HPO4 + NaOH]

Energetics of Calcium Phosphate Nanoparticle Formation

Figure 7. Enthalpy of the reaction Ca(NO3)2 + (NH4)2HPO4 as a function of the concentration of NaOH present in the (NH4)2HPO4 o solution. The solid line refers to the averages of each ∆rHm (hl) and ∆rHom(ll) pair of values in Table 3, corresponding to the baseline relative to the dilution of the Ca(NO3)2 solution. The dashed line corresponds to the equivalent averages for a baseline defined relative to the dilution of the (NH4)2HPO4 + NaOH solution. The error bars represent mean o o (hl) - ∆rHm (ll)]/2. deviations computed as [∆rHm

Figure 8. Calculated enthalpy of the dilution process [(NH4)2HPO4 + NaOH] + H2O as a function of the molar concentration of NaOH present in the (NH4)2HPO4 solution (see text).

+ H2O process, corresponding to an approximately 1:1 dilution, the molar fractions of the H2PO4- (aq), HPO42- (aq), and PO43(aq) species present in the solution entering the calorimetric cell and in the diluted solution exiting from the calorimeter change with the increase of the pH. These molar fractions can be approximately predicted from the pHP values in Table 3 and the dissociation constants of aqueous H3PO4 (pKa1 ) 2.16, pKa2 ) 7.21, and pKa3 ) 12.32).44 When combined with o o (H2PO4-, aq) ) -1296.29 kJ · mol-1,45 ∆fHm (HPO42-, aq) ∆fHm o (PO43-, aq) ) -1277.4 ) -1292.14 kJ · mol-1,45 and ∆fHm kJ · mol-1 45 they lead to the variation of the enthalpy of the o , with [(NH4)2HPO4 + NaOH] + H2O dilution process, ∆dilHm the concentration of NaOH illustrated in Figure 8. Figure 8 o is approximately shows that below [NaOH] ∼ 20 mM, ∆dilHm constant and close to zero. Above that [NaOH] value a sudden o toward more positive values is and progressive rise of ∆dilHm observed. Hence, the baseline contribution becomes significantly more positive for the high range of [NaOH] thus comparatively decreasing the experimental reaction enthalpy relative to an experiment where such a dilution effect is absent, as when the baseline is defined relative to the dilution of the Ca(NO3)2 solution. Besides these baseline effects, several other factors may o contribute to the general aspect of the ∆rHm vs [NaOH] curves in Figure 7, particularly differences in the chemical composition and crystallinity of the nanoparticles present in the final state of the calorimetric process and in the relative concentrations of H2PO4- (aq), HPO42- (aq), and PO43- (aq) caused by the pH change accompanying the reaction. The obtained results suggest,

J. Phys. Chem. C, Vol. 113, No. 14, 2009 5483 however, that the trend should be mainly determined by the chemical composition of the precipitated nanoparticles. As indicated in Table 2, the crystallinity of the products systematically decreases with the increase of [NaOH]. Because the reaction starts by being endothermic at the low end of [NaOH], and the formation of less crystalline products with the same chemical composition should correspond to more positive enthalpies of reaction (less stable final state of the calorimetric process), if crystallinity was the dominant effect, then a o with [NaOH] toward more monotonically increase of ∆rHm positive values ought to be expected throughout the [NaOH] range. This is clearly not the case, and, in fact, an opposite trend is even observed before the minima of the curves in Figure 7. o from the changes in the relative The dependence of ∆rHm concentrations of H2PO4 (aq), HPO42- (aq), and PO43- (aq) that accompany the reaction is also predicted to be small. A calculation analogous to the one described for Figure 8, and based on the values of pHP and pHf in Table 3, indicated that this contribution is always negative and smaller than -3.5 o vs [NaOH] kJ · mol-1. It cannot, therefore, determine the ∆rHm trend depicted in Figure 7. Some insight about the influence of the reaction conditions on the nature of the nanoparticles formed in the course of the calorimetric experiments can be obtained from the analytical characterization of the materials produced in the preliminary studies performed outside the calorimeter (Table 1). Unfortunately the contents in OH-, PO43-, HPO42-, surface-bound water, and lattice water could not be derived from these data. This was due to the fact that the TG experiments did not allow distinguish water resulting from HPO24 decomposition (condensation to diphosphate) from that retained in the solid, thus preventing the determination of the OH- and PO43- contents through a charge balance. Furthermore, an empirical CaaNab(HPO4)c(PO4)6-c(CO3)d(OH)e · nH2O formula of meaningful stoichiometry could not be obtained by combining the results of the TG experiments with those of H elemental analysis. It is nevertheless possible to conclude that the chemical composition of the materials produced in the calorimetric experiments should significantly change as [NaOH] increases. This is demonstrated by the following: (i) the observed increase of the Ca/P molar ratio toward the value typical of stoichiometric hydroxyapatite (1.67; Figure 5); (ii) the increase of the contents in CO32- and surface-bound and lattice water (Figure 2; note that although the plot refers to the total amount of water, as mentioned above, the quantity of HPO42- that decomposes on heating to produce water through reaction 4, should decrease as [NaOH] increases); (iii) the fact that for the two highest [NaOH] studied the incorporation of Na+ in the samples also occurs (Table 1). These variations in chemical composition are reflected by the o with [NaOH] illustrated in Figure significant change of ∆rHm 7. A plausible reason for the minima observed in the curves at [NaOH] ∼ 20 mM (pHP ∼ 10) may be the formation of nanoparticles with a global chemical composition corresponding to mixtures of different phosphate phases above and below those minima. Solid-state NMR evidence46 indicates that nanoparticles produced from the reaction of Ca(NO3)2(aq) with (NH4)2HPO4(aq) at pH ) 10 consist of a crystalline core of stoichiometric hydroxyapatite and a disordered surface layer of Ca8(HPO4)2(PO4)4 · 5H2O (octacalcium phosphate; OCP) with a Ca/P ratio of 1.33. The formation of this type of biphasic nanoparticle, accompanied by a progressive increase of the Ca10(PO4)6(OH)2/Ca8(HPO4)2(PO4)4 · 5H2O molar ratio as the basicity of the (NH4)2HPO4 solution is raised, could explain the monotonical variation of ∆rHom with [NaOH] observed above

5484 J. Phys. Chem. C, Vol. 113, No. 14, 2009 the minima of the curves. In fact, since the enthalpy of the overall calorimetric process must be an approximately linear combination of the enthalpies of the reactions leading to each phase, a continuous increase or decrease of the relative amounts of both phases must lead to a continuous decrease or increase of ∆rHom. The above scenario is also consistent with the observed increase in the Ca/P molar ratio from ca. 1.5 to 1.67 shown in Figure 2 for [NaOH] g 20 mM (pHP g 10). Furthermore, because an amorphous material should be more prone to retain water, the apparent increase of the amorphous character of the nanoparticle materials as the reaction mixture becomes more basic (Table 2 and Figure 4) could also justify the increase in the amount of retained water as [NaOH] increases (Table 1). The existence of minima in the curves of Figure 7 suggests that the materials formed below [NaOH] ∼ 20 mM must correspond to a different combination of calcium phosphate phases or to mixtures of different types of nanoparticles; o with [NaOH] otherwise, a simple monotonic variation of ∆rHm should be observed throughout the [NaOH] range covered by the experiments. This hypothesis is supported by the SEM results (Figure 3). As mentioned above, these show that the sample precipitated without addition of sodium hydroxide to the [(NH4)2HPO4] solution contains rod-like particles which are morphological different from the approximately spherical particles present in all other samples and may therefore correspond to a different phase or phase combination. Conclusions Significant differences in the chemical composition, morphology, and amorphous character of the nanoparticles produced through the reaction between Ca(NO3)2(aq) and (NH4)2HPO4(aq) can be induced, simply by changing the pH of the reactant hydrogen phosphate solution. The well-known tolerance of calcium phosphates to ionic substitutions and their tendency for nonstoichiometry13,47 makes the complete and accurate elemental and structural characterization of the obtained materials very difficult. Nevertheless, the continuous variation of the nature of the solid products with the reaction conditions is clearly reflected by the corresponding enthalpies of reaction determined by flow calorimetry. This method seems, therefore, a very convenient and sensitive tool to assess the reproducible production of calcium phosphate nanoparticles with a specific composition. Acknowledgment. This work was supported by Fundac¸a˜o para a Cieˆncia e a Tecnologia (Project POCTI/199/QUI/35406) and an exchange project of Acc¸o˜es Integradas Luso-Alema˜s (A31/09) with the Deutscher Akademischer Auslandsdienst. Postdoctoral grants from Fundac¸a˜o para a Cieˆncia e a Tecnologia are also gratefully acknowledged by A.L.C.L. (SFRH/BPD/ 35053/2007) and M.L. (SFRH/BPD/26494/2006). References and Notes (1) Lowenstam, H. A.; Weiner, S. On Biomineralization; Oxford University Press:New York, 1989. (2) Mann, S. Biomimetic Materials Chemistry; Wiley-VCH: Weinheim, 1996. (3) Weiner, S.; Addadi, L. J. Mater. Chem. 1997, 7, 689–702. (4) Baeuerlein, E. Biomineralization. From Biology to Biotechnology and Medical Application; Wiley-VCH: Weinheim, 2000. (5) Mann, S. Biomineralization: Principles and Concepts in Bioinorganic Materials Chemistry; Oxford University Press: New York, 2001.

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