1-D AND 2-D DOUBLE HETERONUCLEAR MAGNETIC RESONANCE STUDY OF THE LOCAL STRUCTURE OF TYPE B CARBONATE FLUOROAPATITE.

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1 Magnetic Resonance in Colloid and Interface Science, Nato ASI series II, (eds) J. Fraissard and B. Lapina, Kluwer Academic Publishers, 2002,76, D AND 2-D DOUBLE HETERONUCLEAR MAGNETIC RESONANCE STUDY OF THE LOCAL STRUCTURE OF TYPE B CARBONATE FLUOROAPATITE. H. SFIHI 1 and C. REY 2 1 Laboratoire de Physique Quantique, CNRS FRE 2312, ESPCI, 10 rue Vauquelin, Paris, France. 2 CIRIMAT, CNRS UMR 5085, Institut National Polytechnique, 38 rue des 36 Ponts, Toulouse, France. Abstract The local structure of a type B carbonate fluoroapatite has been investigated by 1-D and 2-D 13 C{ 1 H/ 19 F and 31 P{ 1 H/ 19 F} MAS Nuclear Magnetic Resonance. The results clearly show the existence of two type B carbonated sites and three fluorine sites. 2 One of the CO 3 sites is located near the apatite surface and in very close proximity to strongly adsorbed water. The other type is close to two of the three fluorine sites. 1. Introduction 2- The association of CO 3 ions with apatites is particularly important for understanding the mineral structure of bone. Indeed, carbonate is the third most abundant constituent of bone and tooth mineral after calcium and phosphate, and plays an important role in the maturation and the reactivity of the mineral phase of calcified tissues. Depending on the substitution sites, one distinguishes type B carbonate apatite where some of 2-2- the PO 4 groups are replaced by CO 3 and type A carbonate apatite where the CO 3 are located in apatitic channels generally occupied by hydroxide and/or fluoride ions. Various methods such as molar composition determination [1], X-ray diffraction [1-9], scanning electron microscopy [6], infrared spectroscopy [6-13], Raman spectroscopy [14], electron spin resonance [9,16], 1 H and 13 C solid state nuclear magnetic resonance [17] have been used to investigate the properties, the morphology and the local structure of synthetic and natural (dental enamel and bone) carbonate apatites. The multiplicity of the techniques clearly reflects the complexity of the material. In addition, variability may be observed depending on the synthesis method. For synthetic carbonate apatite, all of the mentioned studies concern carbonate hydroxyapatite (types A and B) in different forms (powders, crystals and films). In order to understand better the structural modifications induced in bone mineral by diseases such as fluorosis [18-21] or by fluoride salts used in the treatment of osteoporosis [21-23], 1-D ( 1 H and 19 F MAS, 13 C MAS, CP-MAS and DCP-MAS) and 2-D 13 C{ 1 H}, 13 C{ 19 F}, 31 P{ 1 H} and 31 P{ 19 F} heteronuclear chemical shift correlation (HetCor) MAS nuclear magnetic resonance were used in this work to investigate the environments of CO 2-3 and of F - ions in a weakly hydroxylated type B carbonate fluoroapatite. Carbonate fluoroapatite constitutes a model compound close to highly fluoridated mineral bones and geological deposits. 2. Experimental 2.1 PREPARATION OF CARBONATE FLUOROAPATITE. 409

2 The carbonate fluoroapatite powder sample was prepared by double decomposition between a calcium nitrate and ammonium phosphate solutions containing fluoride and carbonate ions, according to a precipitation method proposed by Vignoles [24]. In order to observe the 13 C NMR easily, the carbonate ions that we used was enriched sodium bicarbonate (99 % 13 C). The phosphate-carbonate solution (0.43 g of (NH 4 ) 2 HPO 4 ; 0.30 g of NH 4 F, 1.56 g of NaHCO 3 and 5 ml of a 20% ammonia solution in 80 ml of deionized water) was added dropwise with a peristaltic pump into the boiling calcium solution (0.640 g of Ca(NO 3 ) 2,4H 2 O and 5 ml of a 20 % ammonium hydroxide solution in 200 ml of deionized water). The precipitate was filtered, washed with deionized water and dried in an oven at 100 C. The precipitate obtained was characterized by chemical analysis, X-ray diffraction and FTIR spectroscopy. Calcium was determined by complexometry with EDTA [25], phosphorus of phosphate groups by spectrophotometry of phosphovanadomolybdic acid [25], carbonate by coulometry of the carbon dioxide evolved during acid dissolution, and the fluoride was measured with a ion-selective electrode after dissolution of the sample. The chemical composition is reported in table I. TABLE I. Chemical composition of the carbonate fluoroapatite Ca (% weight) P (% weight) CO 3 (% weight) F (% weight) The atomic ratio (Table II) is consistent with the formation of a very carbonate rich fluoroapatite. Table II. Atomic ratios of the carbonated fluoroapatite Ca/P Ca/(P+C) C/(P+C) F/(P+C) The FTIR data (not shown) indicate the formation of a type B carbonate apatite, where carbonate ions replace phosphate ions. Very faint OH - bands are observed at 714 and 691 cm -1, corresponding to the existence of a very low small fraction of OH - groups hydrogen-bonded to fluoride ions. The presence of cation vacancies in this type of apatite where carbonate ions replace phosphate is in agreement with by the Ca/(P+C) ratio which indicates a deficiency of calcium ions. Similarly, the F/(P+C) ratio reveals an excess of fluoride ions (the theoretical F/P value for pure fluoroapatite is 0.33) which has been attributed to the incorporation of F - ions in the oxygen vacancies created by the replacement of phosphate by carbonate groups [24]. However, such a substitution has been questioned [26], and has never been proved by direct determinations. 2.2 NMR SPECTROSCOPY 1 H and 19 F magic angle spinning (MAS) NMR measurements were performed at and MHz with Bruker ASX 500 and ASX 300 spectrometers operating in a static field of 11.7 and 7.1 T, respectively. The two spectrometers are equipped with 4 mm ultra fast (0 18 khz range) spinning probes. For both nuclei, the 410

3 spinning frequency was 15 khz. The recycle delay was 5 s for 1 H and 30 s for 19 F, in accordance with their spin lattice relaxation times. The 1 H and 19 F chemical shifts were referenced to external tetramethylsilane (TMS) and hexafluorobenzene (C 6 F 6 ), respectively. The 13 C MAS 13 C{ 1 H} CP-MAS and DCP-MAS spectra were obtained at MHz (11.7 T) at different contact times in CP-MAS and different reverse times in DCP-MAS. The recycle delay (D1) was 5 s for CP-MAS and 800 s for MAS. The relatively high D1 value in MAS is due to the long 13 C T 1 which is estimated to be 200 s. The spinning frequency was 5 khz. Differential cross polarization (DCP) [27] which was also called cross polarization with polarization inversion (CPPI) [28,29], cross polarization depolarization (CPD) [30] and inversion recovery cross Polarization (IRCP) [31,32] is particularly efficient for resolving overlapping spectra. This method, which is more sensitive than the normal CP, can be used to separate two or more distinct groups (sites or chemical groups) on the basis of the difference (which could very small) in the strengths of their dipole coupling. From the experimental point of view, this is achieved by reversing the phase of the 1 H rf field after a forward transfer or contact time, t c, such that the 13 C polarization is transferred back to 1 H during the reverse time t r. This causes the 13 C spectrum to successively reduce in amplitude, pass through a null, and finally become inverted. The 13 C{ 19 F} CP-MAS spectra were obtained at MHz (7.1 T). The spinning frequency was 5 khz. The 2D 13 C{ 1 H} and 13 C{ 19 F} heteronuclear chemical shift correlation (HetCor) MAS NMR [33-38] measurements were made at MHz (11.7 T) and MHz (7.1 T). The spinning frequency was 12 khz. The 31 P{ 1 H} and 31 P{ 19 F} HetCor MAS NMR measurements were made at MHz (11.7 T) and MHz (7.1 T). The spinning frequency was 12 khz. For all 2D HetCor measurements a contact time of 5 ms was used. This value gives the maximum of polarization as deduced from CP-MAS measurements. Phase sensitive detection was obtained using TPPI phase cycling. For both 13 C{ 1 H/ 19 F} and 31 P { 1 H / 19 F} CP-MAS, the Hartmann-Hahn conditions were achieved on the sample studied. In order to reduce the time of the experiment in 13 C{ 19 F} and 31 P { 19 F} CP-MAS and HetCor, a 90 flip-back-pulse was used to bring the 19 F magnetization along the Zeeman field just after the cross polarization (CP) period. The long 19 F spin lattice relaxation times in the rotating frame (T 1ρ ) make the use of the flip-back-pulse possible. By this means the recycle delay is reduced from 25 s to 4 s. Furthermore, in all CP and HetCor experiments, eight saturation pulses were applied on polarized spins (i.e. 13 C and 31 P) before the 90 pulse on polarizing spins (i.e. 1 H and 19 F). This suppresses the residual signal which could arise from the direct excitation (during the CP period) of the polarized spins. This signal could be strong particularly at very short contact times (0 500 µs). The 13 C and 31 P chemical shifts were referenced to external TMS and to 80% H 3 PO 4, respectively. The other experimental conditions are given in the figures. In this study HetCor [33,34] is used to correlate specific 1 H (or 19 F) and 13 C (or 31 P) with peak positions in the two-dimensional (2-D) NMR spectrum. This method is analogous to conventional CP, except that the 1 H (or 19 F) magnetization is allowed to 411

4 evolve for a period t 1, which corresponds to the first time domain (F 1 ) of a 2-D NMR experiment, before the magnetization is transferred to dipolar-coupled 13 C (or 31 P) in close spatial proximity for the second time domain (F 2 ), during which the signal is detected. This method has been used in various compounds such as silicas and zeolites [35], silica gels [36], aluminosilicates [37], fluorinated γ-alumina [38], in bone and various calcium phosphates [39]. 3. Results and discussion H AND 19 F MAS NMR The 1 H and 19 F MAS NMR spectra are shown in figures 1-a and 2-c, respectively. For comparison the 1 H MAS NMR spectrum of stoichiometric hydroxyapatite [HAp, Ca 10 (PO 4 ) 6 OH 2 ] is also given (Fig. 1-d). a) b) a) c) b) d) c) (ppm/ T M S) Figure 1 : 1 H 1-D MAS NMR spectra a) 1 H MAS. b) 1 H projection of the 2-D 13 C{ 1 H} HetCor MAS. c) 1 H projection of the 2-D 31 P{ 1 H} HetCor MAS. d) 1 H MAS of HAp (ppm / C 6 F 6 ) Figure 2 : 19 F 1-D MAS NMR spectra a) 19 F projection of the 2-D 13 C{ 19 F } HetCor MAS. b) 19 F projection of the 2-D 31 P{ 19 F} HetCor MAS. c) 19 F MAS. The 1 H MAS spectrum contains two main resonance lines : a broad one centered at 5 ppm and a very structured one at 2 ppm. Similar results were reported by Beshah et al. [17] for carbonate hydroxyapatite. The former is assigned to H 2 O which could be in an adsorbed and/or structural form, as previously mentioned [17]. The latter is located in the region of OH groups (0-3 ppm). At least four resonance lines are observed in this domain : two resolved lines at 0.9 and 1.2 ppm and two shoulders at the same chemical shift (0.2 ppm) as that of the 1 H of HAp, and at 2 ppm. As previously reported for various fluorohydroxyaptites, these resonance lines could reflect the different configurations of OH groups in the apatitic channels [40-42,45]. 412

5 The poor resolution observed for these lines could be due to the remaining dipole coupling (mainly 1 H- 1 H, 1 H- 19 F, ) which is not completely averaged by fast MAS. The 19 F MAS NMR spectra shows clearly the presence of two fluoride ion sites represented by two relatively broad resonance lines at 65 and 78 ppm with different intensities. The line at 65 ppm is attributed to fluoride ions located in the apatitic channel, because its chemical shift is very close to that observed in fluorohydroxyapatites [42-47]. Its broadness results mainly from a continuous chemical shift distribution, although 19 F- 19 F, 19 F- 1 H, and to a lesser extent 19 F- 13 C and 19 F- 31 P dipolar couplings could also contribute. This chemical distribution is due to the presence of OH groups in the apatitic channel which leads to different configurations of F - ions [40,41,45]. In addition, the presence of CO 2-3 ions in the lattice could also influence the chemical shift of fluorides located in the channel. In particular, one distinguishes F - ions close to CO 2-3 and those close to PO 3-4. This being said, the principal result revealed by 19 F MAS concerns the fluorine site at 78 ppm. However, the existence of such a site is not surprising considering that the F/(P+C) ratio reveals an excess of fluoride ion in the sample. Its position within the structure is still to be specified and will be discussed in detail later. The peak of weak intensity at 43 ppm is attributed to teflon impurities C CP AND DCP MAS NMR The 13 C MAS NMR spectra obtained at two Larmor frequencies (ν 0 ) are shown in figure 3-a. (a) t c = 10 ms t c = 5 ms t c = 1 ms (ppm / TMS) t c = 0.5 ms (ppm / TMS) Figure 3 : 13 C { 1 H} (left) and 13 C { 19 F} (right) CP-MAS NMR spectra obtained at different contact times,t c. (a) 13 C MAS spectra : (left) ν 0 = MHz ; (right) ν 0 = MHz. At the two frequencies the spectrum consists of a single asymmetric line with maximum intensity at 170 ppm. Similar results were reported by Besha et al. [17] on 413

6 type B carbonate hydroxyapatite. Note that no peak (166 ppm) corresponding to type A carbonate species [17] was detected. The 13 C{ 1 2- H} CP-MAS spectra (Fig. 3) show that the CO 3 located at 170 ppm are less cross polarized than that ppm, whatever the contact time. The carbon cross polarization time constant depends mainly upon the magnitude of the 13 C- 1 H dipoledipole coupling, i.e. the carbon-proton internuclear distance and the number of coupled spins. The 13 C MAS measurements (Fig. 3-a) show that the fraction of 2- CO 3 located at ppm is much lower than that of the carbonates at 170 ppm. Therefore, the fact that the amount of carbon at 170 ppm is underestimated in the cross polarization measurements means that most of them are more distant from the proton, probably close to fluoride ions. In contrast to the 13 C{ 1 H}, the 13 C{ 19 F} CP-MAS spectra at different contact times are very similar to the MAS spectrum. Nevertheless, the MAS spectrum is still much broader than the 13 C{ 19 F} CP-MAS one, whatever the contact time. This means that the CO 3 groups at ppm are weakly cross-polarized by fluoride ions, and that these carbonates are more distant from fluoride ions than those at 170 ppm. More precisely, considering the fact that most of fluoride ions are located in the apatitic channel, this result signifies that the CO 3 groups at ppm will not be located in the bulk but near the surface. These groups correspond to the labile CO 3. In addition and as discussed above, the CO 3 groups at 170 ppm are on average more strongly coupled to the fluoride ions than to the protons. t r = 400 µs t r = 450 µs t r = 500 µs t r = 525 µs t r = 550 µs (ppm/tms) 166 Figure 4 : 13 C{ 1 H} DCP-MAS NMR spectra at different reverse CP times t r, and at contact time, t c = 300 µs ; (NS = 8196). The 13 C CP-MAS measurements raised several questions and did not clearly show whether the CO 3 groups at ppm or a part of those at 170 ppm are more strongly coupled to the protons. In order to elucidate this question, a differential cross polarization (DCP) experiment was performed at different reverse times, t r (Fig. 4). The choice of the contact time which gives the reverse of the 13 C magnetization in DCP measurements is empirical. The value used in this work (t c = 300 µs) is same as that used by Wu et al. [27] to separate the overlapped protonated (HPO 4 ) and unprotonated (PO 4 ) phosphate groups in bone mineral. The DCP shows clearly the existence of two type B carbonate sites characterized by slightly different cross polarization time constants, confirming the qualitative analysis of the 13 C CP-MAS spectra. The amplitude of 13 C magnetization decreases with increasing t r, passes through a null, and becomes negative. The fact that the peak at 170 ppm inverted first means that the cross 414

7 polarization time constant of the corresponding CO 3 groups is slightly shorter than that of the CO 3 at ppm. This means that, at the local level, the former are slightly closer to the protons (more strongly coupled) than the latter. Therefore, the 13 C CP- MAS results cannot be explained by a difference in the distances between protons and each of two types of the CO 3 group, but by a difference in the total number of protons cross polarizing each of them. The total number of protons cross polarizing the labile CO 3 (168.5 ppm) is greater than that of those cross-polarizing the regular type B CO 3 (170 ppm). This means that these protons are different and, as we will see in the next section, they correspond to adsorbed water molecules and to the OH groups in the apatitic channel, respectively C{ 1 H} AND 13 C { 19 F} HetCor MAS NMR The NMR measurements presented in the previous sections, indicated clearly the existence of two main type B carbonate sites. On average, one carbonate (168.5 ppm) is slightly more coupled to the protons than the other (170 ppm), although locally we observed the opposite. However, this other carbonate is strongly coupled to the fluoride ions which are located in two different sites. They also show that the carbonates strongly coupled to the protons (168.5 ppm) are hardly coupled to the fluoride ions (weakly cross polarized by fluorine) H dimension (ppm/tms) F dimension (ppm/c6 F 6 ) C dimension (ppm /TMS) C dimension (ppm/tms) Figure 5 : Contour plots and projections of 2-D 13 C{ 1 H} HetCor MAS NMR. Figure 6 : Contour plots and projections of the 2-D 13 C{ 19 F} HetCor MAS NMR In order to obtain more detailed information on the environment of these different sites (carbonates and fluorine ions), and particularly which carbonate ions are in close proximity to which protons and to which fluoride ions, 13 C{ 1 H} and 13 C { 19 F} HetCor MAS NMR measurements were performed on the sample. Figures 5 and 6 show the contour plots of 2-D 13 C{ 1 H} and 13 C{ 19 F} HetCor MAS NMR and the projections in the F 2 ( 13 C) and F 1 ( 1 H or 19 F) dimensions. The 2-D 13 C{ 1 H}HetCor MAS experiment shows that the water protons (5 ppm) are closer to the CO 3 at ppm than to that at 170 ppm. This is clearly illustrated in Figure 7-a. The 1-D spectrum obtained by summation of the 2-D 13 C row data clearly indicates that the CO 3 groups at are mainly cross-polarized by the protons in the 415

8 range 4-10 ppm (H 2 O). The OH protons (0-2 ppm) practically hardly participate at all in the polarization of these carbonates. However, these protons interact strongly with the CO 3 groups at 170 ppm (Fig. 7-b). This result indicates a heterogeneous distribution of ions and can be interpreted in several ways. It may be interpreted as follows : if the CO 3 at ppm were in the bulk, they should be cross-polarized by the OH protons OH (0-2 ppm) as well as by fluoride ions located in apatitic channel. This is not the case. Therefore, we conclude that these CO 3 groups are close to the apatite surface (labile CO 3 ) and that the water molecules are mainly in the adsorbed form (strongly adsorbed), as mentioned in the previous section. Concerning the OH protons, comparison of the projection in 1 H dimension with the 1 H MAS spectrum (Fig. 1-a and 1-b) reveals that only the OH protons at 0.2 and 2 ppm and appearing as a shoulder in the MAS spectrum participate in the crosspolarization of the CO 3 groups at 170 ppm. This means that the protons at 0.9 and 1.2 ppm are not or are very weakly coupled to both CO 3 sites. Two similar peaks have been reported in octacalcium phosphate by Yesinowski et al. [41]. These peaks were attributed to the protons of water molecules undergoing rapid reorientation on the NMR time scale [41]. This attribution would be fully consistent with the fact that the 0.9 and 1.2 ppm protons were neither cross-polarized by the 13 C of both CO 3 sites nor by the 31 P of PO 4 (see below). Nevertheless, these water molecules are, according to these authors, located in isolated sites in the structure of OCP. In our case the water would be located at the surface (weakly adsorbed) rather than in the structure. We assign the OH protons at 0.2 and 2 ppm to OH. OH (HAp) and to OH. F configurations in the apatitic channel, respectively, as previously reported for fluorohydroxyapatites containing different amounts of fluoride ions [41]. This assignment is corroborated by the relative intensities of the corresponding peaks, which are in good agreement with the chemical data. Indeed, the amount of fluoride ion is greater than that of OH - and therefore the OH. F configurations should be predominant. In addition, the downfield location of the corresponding peak (2 ppm instead of 0.2 ppm) could be explained by strong hydrogen OH. F bonds. The most important result revealed by the 2-D 13 C{ 19 F} HetCor MAS experiment concern the two fluoride ion sites and particularly the site located at 78 ppm. Both types of fluoride ion are coupled to the carbonate species, located at 170 ppm (see also Figures 7-c and 7-d), as expected. However, the relatively high intensity of the peak corresponding to fluoride ions at 78 ppm, compared to that observed in the 19 F MAS spectrum (Fig. 2-c-right), means that these fluoride ions are more strongly coupled to the carbonate ions than those located at 65 ppm (fluoride ions in the apatitic channel). Indeed, they are better cross-polarized. At 500 µs contact time (result shown), the peak at 78 ppm is twice as intense as that at 65 ppm. These results indicate that the fluoride ions corresponding to the former peak are closer to carbonate ions at 170 ppm than those corresponding to the latter. These fluoride ions are probably located at the oxygen vacancies created by the replacement of phosphate groups by carbonate groups (see Fig. 8-left). The replacement of phosphate by carbonate creates a negative charge deficit which is compensated by the creation of a vacancy in a nearby calcium site and a vacancy in a close monovalent ion site (OH - or F - ) [1,4,24], as shown in figure 8-left. These carbonate ions are thus far from F - and OH - ions, and 416

9 a) b) c) d) (ppm/tms) Figure 7 : 13 C NMR 1-D spectra obtained by summation of the 2-D 13 C HetCor/MAS row data corresponding to the CO 3 interacting mainly with protons in range : 10-4 ppm (a) ; 2-0 ppm (b) and with fluoride ions in range : ppm (c) ; ppm (d). the existing ionic vacancies could be occupied by water molecules. This assumption can, however, be rejected, because in this case the protons of the water molecules, which would be located in monovalent (OH - or F - ) or oxygen vacancy sites, should be more strongly coupled to the carbonate ions at ppm than the OH protons located in the channel to the carbonate ions at 170 ppm. This is not the case, as is revealed by the DCP-MAS measurements (the cross polarization time constant of the carbonate ions at ppm is slightly shorter than that of those at 170 ppm). The water molecules would be mainly in the adsorbed form. However, on the contrary, if a phosphate is replaced by CO 3,F association (Fig. 8-right), as previously mentioned [1,4,24], although such a substitution has been questioned [26], there is no local charge deficit and the cationic and anionic vacancies do not form. The fluoride ion (F 3 site in fig. 8-right) in the CO 3,F association, which may correspond to the peak at 78 ppm in the 19 F MAS spectrum, should be slightly more strongly coupled to the corresponding CO 3 than the fluoride and/or OH - ions located in the apatitic channel (F 2 site). Another point confirming the attribution of the peak at 78 ppm to the F 3 site is its relative intensity. From chemical data the amount of CO 3,F which corresponds to the fluoride ion excess, would be close to approximately 30 % of the total amount of fluoride. This value is consistent with the relative integrated intensity of the peak at 78 ppm deduced from a rapid peak fitting of the 19 F MAS spectrum (result not shown). The peak corresponding to the fluoride ions located in the apatitic channel (65 ppm), appears only as a shoulder in the projection in the 19 F dimension of the 2-D HetCor measurements (Fig. 1-a). In addition to the shoulder we notice another unresolved peak at 70 ppm. The overall intensity of both peaks is dominated by that of the peak at 70 ppm. Although both peaks correspond, as previously indicated, to fluoride ions in the apatitic channel, this result reveals that the fluoride ions at 70 ppm are more strongly coupled to the carbonate ions at 170 ppm, than those at 65 ppm. We attributed the latter to the F 1 site (Fig. 8-right). It corresponds mainly to fluoride ions in a phosphate environment. The peak at 70 ppm, which we assign to fluoride ions in a carbonate environment (F 2 site), should also be also present in the 19 F MAS spectrum. That this peak did not appear clearly in this spectrum can be explained by the fact that the number of PO 4 (hence the number of fluoride ions close to PO 4 ) is greater 417

10 than that of the CO 3 (hence the number of fluoride ions close to CO 3 ). Chemical analysis indicates that only 20% of the phosphate sites were replaced by a carbonate ion. This is in good agreement with the fact that the intensity of the 19 F MAS spectrum is dominated by the peak centered at 65 ppm (F 1 site) compared to that of the peak at 70 ppm (F 2 site). II P C 3- PO 4 F 1 Ca Z=3/4 O (or OH) Vacancy F III III III' III' 2- CO 3 CO 3 II F 2 Z=1 / 4 I I (or OH) PO 3-4 F 3 Figure 8 : Site occupancy of fluoride ions in carbonate fluoroapatite as revealed by NMR. Another interesting result revealed by the 2-D 13 C{ 19 F} HetCor MAS experiment is the existence of a third fluoride ion site. This site, which is represented by the peak at 90 ppm in the 19 F spectrum obtained by the projection in the 19 F dimension of the 2- D HetCor measurements, is at the same chemical shift as that of ammonium fluoride (NH 4 F). This ammonium fluoride is an impurity of the synthesis and could be located on a calcium-deficient site, as previously mentioned [9,48]. This perfectly explains the cross-polarization of the NH 4 F fluoride ions by the CO 3 carbon and vice versa. However, the amount of NHF 4 remains very small since no significant signal corresponding to this species was detected by 19 F MAS (Fig. 1-c). This means that all the oxygen vacancies would not occupy by F 3 sites, as discussed above, but some of them (same amount as that of NH 4 F which is very weak) remain unoccupied. It is the same for the vacancies at the monovalent ion site (F - 2 or OH - ). Similarly, this result suggests that the projection in the 1 H dimension of the 2-D 13 C{ 1 H} HetCor MAS measurements (Fig. 1-b and 5-left) contains a contribution of NH 4 F whose protons resonate at 7.2 ppm P{ 1 H} AND 31 P { 19 F} HetCor MAS NMR The contour plots of 2-D 31 P{ 1 H} and 31 P { 19 F} HetCor MAS NMR and the projections in the F 2 ( 31 P) and F 1 ( 1 H or 19 F) dimensions are shown in figures 9 and 10, respectively. The 2-D 31 P{ 1 H} HetCor (contours and projections) shows that the PO 4 groups are connected to the same protons as the two type B CO 3. However, the 418

11 peak at 0.2 ppm, which we assigned to OH in OH OH configurations, appears more clearly in the projection in the 1 H dimension. Furthermore, the intensity of both peaks (0.2 and 2 ppm) is higher than that observed for the same peaks obtained by the projection in the 1 H dimension of the 2-D 13 C { 1 H} HetCor (see figures 1-b and 1-c). This is explained by the fact that there are more PO 4 groups than CO 3 species. The protons of water molecules seem to be less cross-polarized by phosphorus than by the labile CO 3, although the number of PO 4 groups is greater than that of the latter species. Furthermore, the peak intensity of these water molecules contains a contribution from NH 4 F whose protons resonate at 7. 2 ppm. This means that the PO 4 groups are more distant from the water molecules than the labile CO 3, confirming then that the water molecules are adsorbed (strongly) at the surface. We could also note that the peaks at 0.9 and 1.2 ppm observed in the 1 H MAS spectrum and previously discussed in detail are absent from the projection in the 1 H dimension (Fig. 1-c and 9). This confirms the attribution of the two peaks to water molecules undergoing rapid reorientation at the apatite surface H dimension (ppm/tms) F dimension (ppm/c6 F 6 ) P dimension (ppm/h 3 PO 4 ) Figure 9: Contour plots and projections of the 2-D 31 P{ 1 H} HetCor MAS NMR P dimension (ppm/h 3 PO 4 ) Figure 10 : Contour plots and projections of the 2- D 31 P{ 19 F} HetCor MAS NMR. The surprising result in the 31 P { 19 F} HetCor is that the projection in 19 F dimension leads to a spectrum very similar to that obtained by 19 F MAS. This means that the fluorine sites at 78 and 65 ppm are practically at the same distance from the PO 4 groups. This is a good agreement with a location of the fluoride ions at 78 ppm at oxygen vacancy sites (F 3 sites), confirming then the interpretation of the 2-D 13 C{ 19 F} HetCor measurements. 4. Conclusion We have shown in this work that the combination of 1-D and 2-D double nuclear magnetic resonance involving different spins ( 1 H, 19 F, 13 C, 31 P) can provide more detailed information on the local structure of carbonate fluorohydroxyapatites, and more generally on materials of complicated structure as bone minerals. 419

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