Synthesis and Characterization of Nanoapatites Organofunctionalized with Aminotriphosphonate Agents

Abstract

Organofunctionalized apatite nanoparticles were prepared using a one step process involving dissolution/precipitation of natural phosphate rock and covalent grafting of nitrilotris(methylene)triphosphonate (NTP). The synthesized materials were characterized by Brunauer–Emmett–Teller (BET) surface measurement, thermogravimetry, inductively coupled plasma emission spectroscopy (ICP-ES), elemental analysis, multinuclear solid state cross-polarization/magic angle spinning (CP/MAS) and single-pulse NMR spectroscopy, transmission electron microscopy (TEM) and energy dispersive x-ray analysis (EDXA). After grafting BET measurements yielded particle specific surface areas ranging from 88 to 193 m² g⁻¹ depending on the grafted phosphonate. The results show that the surfaces of the nanoapatite particles can be covered with functional groups bound through a variable number of R-P-O-Ca bonds to render them organoapatites.

1. Introduction

Apatites are orthophosphates exhibiting a rich, highly complex, often confusing chemistry, the term “apatite” being derived from the Greek απαταω, meaning “fraud” or “deceive”. Hydroxyapatite Ca₁₀(PO₄)₆(OH)₂ (HAp) is a naturally occurring mineral widely used in development of bioceramics, environmental sorbents and heterogeneous catalysis [1-6]. In terms of composition, the mineral resembles the nanocrystals embedded in the collagenous matrix in all vertebrate bone and in the dentin and enamel of teeth; the surface chemistry of bone nanocrystals has important implications for both bone biology and for the action of pharmaceuticals used to treat metabolic bone disease [7]. Thus, apatite interfacial chemistry and the derivatization of apatite nanocrystal surfaces have widespread relevance to many areas in chemistry, biology, materials science, geochemistry, environmental remediation and medicine.

In recent years, many studies have demonstrated that a wide range of additives, such as silane [8] and titanate-coupling agents [9-10], biomacromolecules [11] and polymers [12] can modify the structure and morphology of apatite and control its nucleation. Above all, a number of studies have been carried out to elucidate the effect of various organic molecules on the crystallization of hydroxyapatite. On the other hand, phosphonate additives have important applications in the biomineralization process [13], most importantly to control the remodeling of bone in the treatment of osteoporosis and other diseases of bone metabolism [14]. The usefulness of organophosphonates is enhanced by their stability over wide ranges of pH and temperature. Thus, several works were carried out on the preparation of hybrid apatites demonstrating the influence of small organic ligands on structural and thermal properties, knowing that the organic species are among the most effective inhibitors of the crystal growth of calcium phosphates [15]. Tanaka et al. [16] succeeded in grafting monohexylphosphate and monodecylphosphates. Other studies were devoted to the morphological control of apatite growth, for example, through a polymeric route using calcium nitrate and phenydichlorophosphine C₆H₅PCl₂ as starting materials enabling the formation of hydroxyapatite layers [17]. El Hammari, et al. [18] prepared organohydroxyapatite products via a rapid precipitation method by reacting Ca(OH)₂ and NH₄H₂PO₄ in the presence of phenyl and alkyl phosphonate species separately. The latter results indicate that the phosphonate introduced into the apatite lattice has a strong effect on its specific surface area and porosity. In addition, Silva et al.⁸ demonstrated that silylation of apatite by (CH₃O)₃SiR agents affects the surface properties and therefore heavy metal adsorption by the presence of the amine function in the organic surface group. Colloidal synthesis and characterization of a crystalline apatite hybrid with aminoethylphosphate ligands has been achieved in ref. [19], demonstrating that ultrafine individualized calcium phosphate nanophosphors displaying amino groups on their surfaces are good candidates for use as fluorescent probes in biological imaging. However, the high reactivity of apatites with organic phosphorus molecules resides in the presence of charges and hydrogenous active sites on their surfaces, in particular the amphoteric sites ≡Ca-OH and ≡P-OH [20]. The presence of these hydrogenous sites enables reaction with organic additives to form hybrid apatites [21], whereas organic surface moieties can change the surface chemical (hydrophilicity, hydrophobicity, reactivity) properties of the solid, as well as structural and physical properties.

It is well known that nanoapatite powders can be produced using different methods such as sol-gel [22], hydrothermal [23] or precipitation methods [24-25], through a double decomposition reaction in aqueous solution between Ca(NO₃)₂ and (NH₄)₂HPO₄, or by a neutralization reaction in which Ca(OH)₂ is neutralized by H₃PO₄ in an aqueous slurry. Up to now, the precipitation route is often employed in industrial hydroxyapatite synthesis because of the availability of low cost raw materials, as well as its relative simplicity. There are several investigations on the preparation of nanosized apatites under optimal conditions, illuminating the influence of temperature, pH and the presence of surface modifiers on the size and phase composition of the obtained material [23-26].

In this work, hydrophobic hydroxyapatite particles with structural features in the range below 100 nanometers were synthesized in situ in aqueous solution in the presence of nitrilotris(methylene)triphosphonates (NTPs) using calcium and phosphorus precursors from Moroccan natural phosphate. The present investigation explores a novel synthesis and characterization of hybrid mesoporous apatite resulting from the simultaneous reactions of the dissolution of natural phosphate and the precipitation of mesoporous apatite in the presence of nitrilotris(methylene)triphosphonates (NTP) species. With a one-step grafting process, multifunctional molecules containing phosphonate and nitrogen groups with a reactive centre are formed and linked to the apatite surface. In a companion paper, we describe a first environmental application using these new organo-structured mesoapatites for removal of lead from aqueous solution. The ability to modify an apatite surface with specific ligand fields creates powerful new capabilities for other applications such as catalysis, chemical separations, and sensor development.

2. Experimental

2.1 Sedimentary phosphate rock

Phosphate rock samples used here come from an extracted ore of Bengurir (Morocco). Prior to use, this material requires initial treatments such as crushing and washing. The 100–400 μm grain size fraction was washed with distilled water several times to remove the soluble matter. As published elsewhere [27], the chemical composition of the natural phosphate was determined as: Ca (37.84%), P (15.03%), F (3.07%), Si (1.78%), S (0.78%), Na (0.79%) and other negligible elements. The structure and morphology of the phosphate rock sample was estimated using different characterization techniques. X-ray diffraction indicated the presence of two main inorganic components (apatite + quartz-SiO₂), therefore solid state NMR spectroscopy was used to identify the environments of phosphorus, silicon and carbon in the Bengurir natural phosphate. Thermal analysis showed that small quantities (7 wt. %) of organic components were still present [27].

2.2 Synthesis of grafting minerals

All chemicals of reagent quality were obtained from Aldrich Chemical Co. Nitrilotris(methylene)triphosphonic acid was used as a commercial 50 wt.% solution in water. The preparation of grafted apatites followed the same process described in a previous study [28], except that the NTP phosphonate was introduced in (Ca+P) solution: a phosphate rock (PR) mass of 20.6 g was dissolved in 500 ml of deionized water adjusted to pH=2 with 1M HNO₃ solution and then stirred for 3 hours. After dissolving, the solution was filtered and the filtrate was mixed under vigorous stirring with nitrilotris(methylene)triphosphonates (NTP) in varying proportions. The transparent mixture was precipitated using 20 mL of concentrated ammonia solution at pH =10. The final white suspensions were aged for 24 h at room temperature, filtered, washed with deionized water and then dried overnight at 100 °C. The sample labels (e.g., x% NTP) refer to the molar ratio organic phosphorus/(organic + inorganic phosphorus).

2.3 Techniques

The crystalline phases were identified using a Philips PW131 powder X-ray diffractometer (XRD) using a copper source. Infrared spectra were recorded from 400 cm⁻¹ to 4000 cm⁻¹ on a Bruker IFS 66v Fourier transform spectrometer using KBr pellets. The N₂ adsorption-desorption isotherms for dried powders were obtained by multi-point N₂ gas sorption experiments at 77 K using a Micromeritics ASAP 2010 instrument. The specific surface areas were calculated according to the Brunauer–Emmett–Teller (BET) method using adsorption data in the relative pressure range from 0.05 to 0.25, whereas the pore size and volume were estimated using the Barret–Joyner–Halenda (BJH) approximation. Thermogravimetry (TG) was carried out in flowing air using a TA Instruments Netzsch STA-409EP apparatus. Samples were initially dried at 100 °C. Thermal measurements were conducted from 30°C to 1000°C at a 10°C/min heating rate. The sample powder was chemically analyzed by inductively coupled plasma (ICP) emission spectroscopy (ICPS-7500, Shimadzu, Japan) and elementary CNHOS analyses. Solid-state ¹³C and ³¹P NMR spectra were obtained at frequencies of 75.5 and 121 MHz, respectively, using single 90° pulse magic angle spinning (MAS) measurements on a Bruker 300 MHz spectrometer.Transmission electron microscopic (TEM) analysis with energy dispersive (EDXA) detection was performed on Cu-coated carbon grids using a high resolution thermal field emission electron microscope equipped with an EDXA system for elemental analysis (NEKAI-G² environmental transmission electron microscope) operating at 100kV.

3. Results and discussion

3.1 Characterization of grafted apatite

XRD patterns of the as-prepared products showed a poorly crystalline apatite structure, the crystallinity of which strongly depended on the NTP concentration (Fig. 2). This reveals the presence of very small particles and a significant degree of disorder in the apatite lattice caused by the presence of NTP molecules, which inhibit apatite crystallization. The structural disorder induced by NTP incorporation is responsible for the observed reduction of the thermal stability of the grafted materials. In fact, heat treatment of the powders induces a partial conversion of NTP-HAp particles into β-tricalcium phosphate β-Ca₃(PO₄)₂ (β-TCP), the extent of this conversion increasing with the incorporated NTP content. However, for environmental applications, i.e., using the grafted products as adsorbents, no heat treatment is recommended to conserve the surface properties and the chemical composition of the materials.

Fig. 2.

Change in XRD patterns of NTP-HAP treated with various NTP agents as received and after heating, compared to that of the phosphate rock (PR) starting material.

Infrared spectra of the various products display the vibrational modes of PO₄ groups at 1100, 1050, 960, 605 and 564 cm⁻¹ characteristic of the apatite structure (Fig. 3). The small displacement of PO₄ bands to high frequencies with NTP content is related to the structural disorder and the nature of the P-O bond [29]. However, the P-O bands from organic phosphorus PO₃²⁻ in NTP molecules overlap those of inorganic PO₄³⁻ ions in the apatite lattice. The FT-IR spectra of grafted NTP-HAp composites exhibit weak peaks originating from organic carbon in the wavenumber range of 1420–1450 cm⁻¹, contrary to the ungrafted materials, which are carbonated apatites. This reflects the replacement of CO₃²⁻ ions originally in the phosphate rock with phosphonate groups in the grafted materials. Strong carbonate bands are found in both the starting PR material and in the reference sample prepared without NTP species. After surface grafting, new vibrational bands appear at 2000, 2300 and 2900 cm⁻¹ belonging to the aliphatic C-H, N-C and C-P stretch of NTP in the apatite lattice [30].

Fig. 3.

FT-IR spectra of NTP-HAP powders with various NTP concentrations as-received and after heating at 800°C, compared to that of the phosphate rock (PR) starting material.

In order to obtain further information on the conformation and the bonding nature of phosphonate molecules in the apatite structure, ³¹P and ¹³C MAS-NMR measurements were performed. From ³¹P MAS-NMR analysis of ungrafted apatite, a single phosphorus peak is observed at 2.9 ppm versus 85% H₃PO₄ similar to that found in synthetic apatites and bone and teeth [31-33]. This confirms that one crystallographic site is available in the structure as demonstrated in previous works [34-36]. In addition to the sharp resonance at 2.9 ppm, one broad resonance around at 19.6 ppm is observed in grafted apatite (Fig. 3) characteristic of organic phosphorus in NTP species linked to calcium ions because they have been either adsorbed onto the apatite surface or incorporated into the apatite phosphate structure. We have shown that NMR can be a helpful adjunct to XRD in ascertaining the state of the material. In the examination of the grafted samples, the XRD results indicate the presence of a single apatite phase but give no indication for any remaining organic material or of the relative amounts. ¹³C NMR spectroscopy has identified the carbon environments associated to the organic matter from NTP molecules at 60 ppm and has shown a small quantity of residual carbonate in an apatitic environment at 160 ppm, especially for the HAp reference. Other experiments are now in progress to identify the structural change with heating treatment of grafted samples and to quantify their distribution between the possible sites in the crystal lattice.

3.2 Thermal analyses and stability

The TG curves for the as-received products with and without NTP substrates are presented in Fig. 6. It can be obviously seen that a larger weight loss occurs in the temperature range of 200 to 600 °C, which is attributed to the thermal decomposition of organic substrates in the apatite powders. We can observe that the weight loss of the products increases with the grafted NTP content. Another stage is from 600 to 1000°C, where the small mass loss was observed, assigned to the departure of the water during structural transformations [37]. We note that the ungrafted apatite has a higher total mass loss than that produced by the modified apatite with 2.5 wt.% NTP. We hypothesize that a major part of the weight loss in the ungrafted material comes from water desorption. It is known that native HAp has a hydrophilic surface and absorbs a large amount of water. The DTA thermograms of the grafted apatites are given in Fig. 7. One endothermic peak is observed at 120°C, attributable to the desorbed water molecules. The first exothermic peak, beyond 380°C, corresponds to the combustion of the organic nitrogenous fraction of the sample. Finally a second exothermic peak at 700°C is related to the transformation of the apatite phase to β–Ca₃(PO₄)₂ following the total combustion of the organic matter.

Fig. 6.

TG analyses of pure and NTP-grafted apatites.

Fig. 7.

DTA thermograms of pure and NTP-grafted apatites.

Chemical analyses of ungrafted and grafted apatites are summarized in Table 1. The synthesis conditions greatly influenced the composition of the grafted HAp powders. With unvarying Ca and P content of the phosphate rock starting material, the added NTP molecules could be differentiated on the basis of the Ca/P molar ratio of the precipitates. For high NTP content, the Ca/P values are close to 1.5, characteristic of β-Ca₃(PO₄)₂ detected by XRD for NTP-grafted HAp thermally treatment at 800 °C. This confirms the accuracy of the chemical analyses.

Table 1.

Chemical composition and porous characteristics of pure and graft-modified apatites: elemental analysis, calcium/phosphorus molar ratio, thermogravimetric weight loss, specific surface area S_BET, specific pore volume V_p, mean pore diameter D_p, and lead (II) adsorption capacity q_max.

Solid	%Ca	%P	%C	%N	Ca/P	Wt. loss % 200-600°C	SBET (m2 g−1)	Vp (cm3 g−1)	Dp (nm)	qmax (mmol g−1)
PR							20			0.48
0 % NTP	38.66	15.33	0.30	-	1.95	2.02	150	36.2	11.5	1.68
2.5 % NTP	34.31	16.24	0.52	0.20	1.61	3.49	193	45.2	9.5	2.13
5 % NTP	34.41	17.10	0.72	0.32	1.55	4.62	148	35.5	25	2.97
10 % NTP	34.39	17.39	1.05	0.62	1.53	6.03	145	33.3	38	3.06
20% NTP	34.01	17.05	1.65	-	1.52	7.10	88	20.2	40	-

3.3 Textural properties

Nitrogen adsorption-desorption on grafted apatite resulted in a type IV isotherm according to Brunauer’s classification [38] (Fig. 8). Isotherms exhibit a hysteresis loop which indicates that the grafted samples act as mesoporous materials. As presented in Table 1, the BET specific surface area was 193 m²g⁻¹ for the 2.5% NTPA-grafted apatite, compared to 150 m²g⁻¹ for the ungrafted apatite and to 20 m²g⁻¹ for phosphate rock. Modification by NTP increased the surface area of apatite only at the lowest NTP content; increasing the NTP content resulted in a gradual reduction of surface area at 145 m² g⁻¹ for 10% NTP content. This is related to the high molecular disorder introduced by the organic molecules in the apatite structure; it was therefore possible that organic matter is located in pores or blocks the pore entrances [30]. Pore size distributions (Fig. 9) indicated the diameter of pores in the functionalized apatite were between 9.5 and 40 nm, including mesopores.

Fig. 8.

N₂-sorption isotherms at 77 K for pure and NTP-grafted apatites.

Fig. 9.

Pore size distribution from BJH analysis for pure and NTP-grafted apatites.

NTP-modified apatite powders were difficult to image by SEM due to the presence of organics. All SEM images mainly showed featureless granular materials, with micron-size platelets being sometimes visible. TEM studies indicated that the materials consist of aggregations of particles; the particles exhibit dimensions well below 100 nm (Fig. 10). Noticeably, 2.5% NTP and 5% NTP showed a rather open structure with individual particles being easy to distinguish, whereas the density of aggregation appeared to increase at 10% NTP. Other particles appeared to consist of barely individual nanoparticles held together by an amorphous, probably organic matrix. The size of the individual NTP-HAp particles in the aggregates ranges from 40 to 90 nm.

Fig. 10.

TEM images of apatite grafted with NTP molecules.

4. Conclusions

New organofunctionalized apatites were synthesized from natural phosphate rock by grafting aminotrimethylene phosphonic acid by means of a low-cost process. The introduction of these organic moieties into the apatite lattice most importantly provides an opportunity to control the nanocrystal surface properties that may prove useful in a number of applications. In particular, the introduction of amine groups provides the potential for complexation of metal ions in environmental applications such as the removal of lead from water.

Fig. 1.

Structure of nitrilotris(methylene)triphosphonic acid.

Fig. 4.

³¹P-NMR spectra of NTP-grafted HAp and pure HAp as reference (0% NTP). The peaks at ±70 ppm are rotational sidebands (artifacts resulting from the spinning and not actual resonances).

Fig. 5.

¹³C-NMR spectra of NTP-grafted HAp and pure HAp as reference (0% NTP).