**3.1.9. Nuclear magnetic resonance**

efficiency is greater than that for NR-laser-SNMS, the quantification is also simpler and extremely high selectivity prevents almost all isobaric and molecular interferences [78],[79].

120 Apatites and their Synthetic Analogues - Synthesis, Structure, Properties and Applications

The method of NR-laser-SNMS was used by DAMBACH et al [80] to investigate different states of biomineralization in vitro. The results indicate that in the vicinity of single osteoblasts, extracellular enrichment of potassium typically occurs during initial stages of mineralization. Potassium may interact with matrix macromolecules and prevent an uncontrolled apatite deposition. However, apatite biomineral formation is correlated with a potassium release. In conclusion, potassium seems to be involved in the process of extracellular matrix biomineral‐

The concept of electron paramagnetic resonance (EPR) spectroscopy is very similar to more familiar nuclear magnetic resonance (NMR). Both methods deal with the interaction be‐ tween electromagnetic radiation and magnetic moments. In the case of EPR, the magnetic moments arise from electron rather than nuclei. The term EPR was introduced as a designa‐ tion taking into account contributions from electron orbital as well as spin angular momen‐ tum. The term electron spin resonance (ESR) was also widely used because in most cases the absorption is linked primarily to the electron-spin angular momentum [82],[83]. EPR spec‐ trum is a diagram in which the absorption of microwave frequency radiation is plotted against

The technique of electron paramagnetic resonance spectroscopy may be regarded as the consequence of the STERN–GERLACH experiment. They showed (in 1920) that an electron magnetic moment in an atom can take on only discrete orientation in a magnetic field, despite the sphericity of the atom. Subsequently, UHLENBECK and GOUDSMIT liked the electron magnet‐ ic moment with the concept of electron spin angular momentum. In hydrogen atom, there is additional angular momentum arising from the proton nucleus. BREIT and RABI described the resultant energy levels of hydrogen atom in a magnetic field. RABI et al [81] studied the transition between levels induced by an oscillating magnetic field, and this experiment was the first observation of magnetic resonance. The first observation of electron paramagnetic resonance peak was made in 1945 by ZAVOISKY, who detected the radiofrequency absorption

line from CuCl2·2H2O sample using the radiofrequency (RF) source at 133 MHz [82].

The major components of EPR spectrometer are shown in **Fig. 6**. The microwave bridge supplies the microwaves at controlled frequency and power, which are transmitted to the sample cavity via the waveguide. The sample cavity is placed perpendicular to applied magnetic field, which can be varied in controlled way. In addition to this main magnetic field, a controlled but smaller oscillating magnetic field is superimposed on the cavity via the Zeeman modulation frequency. The ideal way to perform the experiment would be to apply a fixed magnetic field and vary the microwave frequency. However, microwave generators are only tunable over very limited ranges. Thus, the microwave frequency is fixed and applied magnetic field is varied. The magnetic field is applied until it reaches the value at which the sample will absorb some of the microwave energy, i.e. and EPR transition occurs [84],[85].

ization.

**3.1.8. Electron paramagnetic resonance**

the magnetic field intensity [83].

Solid-state nuclear magnetic resonance (NMR) is a technique for accurate measurement of nuclear magnetic moments where the resonance frequency depends on its chemical environ‐ ment [97],[98],[99]. The method can provide useful information on the number of molecules in the asymmetric unit and on the site symmetry of the molecule in the lattice to assist in the refinement of powder X-ray diffraction (**Section 3.1.1**) data. The method can distinguish between different polymorphs. Alternatively, solid-state NMR can be used for direct and accurate measurement of internuclear distances. For amorphous and disordered solids, such as inorganic glasses and organic polymers, solid-state NMR provides structural information that cannot be obtained by any other technique [100],[101]. NMR is also the diagnostic method used in veterinary science and medicine particularly in clinical research of human brain by magnetic resonance imaging (MRI) [102].

The solution-state NMR method was developed for the investigation of structure of soluble proteins [103]. Solution and solid-state NMR are both excellent methods for the determina‐ tion of chemical composition [100].

The structural information of apatites is usually investigated from 1 H, 19F and 31P NMR spectra of apatites [104]. The 31P solid-state NMR spectroscopy is a useful tool to investigate structur‐ al information about apatites on bone organic and inorganic mineral components, as well as to investigate the crystallinity and compositional changes in carbonated apatites [105]. Intact bone is a demanding tissue for structural studies. Serious experimental problems arise from the morphological diversity of bone and from the co-existence, interrelationship and great complexity of its organic and inorganic components. Furthermore, one has to perform noninvasive analysis because bone samples are very sensitive to physical effects and chemi‐ cal treatment. Solid-state 31P NMR gives us a unique opportunity to look specifically at the minerals of whole bone without any chemical pretreatment, thus avoiding the intervention into the bone structure [106].

**Fig. 7.** Nuclear separation along parallel chains (the crystallographic c-axis) in various apatites (a): FFF group (I), FFH group (II) and HFH group (III). Correlation between observed 19F line width and fluorine content of fluorinated hydroxylapatite (b) [104].

The 19F NMR spectrum of fluorinated calcium hydroxylapatite (Ca10(PO4)6F2*x*(OH)2–2*x*, where *x* is the fraction of OH<sup>−</sup> replaced by F<sup>−</sup> ) indicates the correlation between 19F chemical shift tensor parameters and the content of fluorine in apatite. The presence of OH<sup>−</sup> groups induces perturbations of fluorine environments, involving the displacements of both fluorine and hydroxyl groups from their normal positions. This leads to a distortion of the electronic environment with regard to the investigated fluorine nucleus and gives reasons for ob‐ served change in the 19F chemical shift tensor of fluoridated hydroxylapatite with different fluorine content. Furthermore, the presence of OH-group destroys the fluoride long-range structure and that results in an isotropic chemical shift distribution. This leads to observed increase in the 19F line width in the case of low fluorine content [104],[107].

#### **3.1.10. Scanning electron microscopy, structure and elemental analysis**

The scanning microscope (SEM) permits the observation and characterization of heterogene‐ ous organic and inorganic materials on a nanometer (nm) to micrometer (μm) scale. In SEM, the area to be examined or the volume to be analyzed is irradiated with finely focused electron beam, which may be swept in a raster across the surface of the specimen to form an imager or may be static to obtain the analysis at the position. The type of signals produced from the interaction of the electron beam (primary electron, PE) with the sample (**Fig. 8**(**a**)) includes secondary electrons (SE, with energy ≤50 eV), backscattered electrons (BSE, *E* > 50 eV), Auger electrons (AE), X-ray characteristics (X) and other photons of various energies such as continuum X-rays and heat. Low-loss electrons (LLE) show the energy losses of a few hundreds of eV. These signals are obtained from specific emission volumes within the sample and can be used to examine many characteristics of the sample such as surface topography, crystal‐ lography, composition, etc. [108],[109],[110].

**Fig. 8.** Electron–specimen interaction (a) and schematic energy spectrum (a) [109].

The structural information of apatites is usually investigated from 1

122 Apatites and their Synthetic Analogues - Synthesis, Structure, Properties and Applications

into the bone structure [106].

hydroxylapatite (b) [104].

is the fraction of OH<sup>−</sup>

replaced by F<sup>−</sup>

of apatites [104]. The 31P solid-state NMR spectroscopy is a useful tool to investigate structur‐ al information about apatites on bone organic and inorganic mineral components, as well as to investigate the crystallinity and compositional changes in carbonated apatites [105]. Intact bone is a demanding tissue for structural studies. Serious experimental problems arise from the morphological diversity of bone and from the co-existence, interrelationship and great complexity of its organic and inorganic components. Furthermore, one has to perform noninvasive analysis because bone samples are very sensitive to physical effects and chemi‐ cal treatment. Solid-state 31P NMR gives us a unique opportunity to look specifically at the minerals of whole bone without any chemical pretreatment, thus avoiding the intervention

**Fig. 7.** Nuclear separation along parallel chains (the crystallographic c-axis) in various apatites (a): FFF group (I), FFH group (II) and HFH group (III). Correlation between observed 19F line width and fluorine content of fluorinated

The 19F NMR spectrum of fluorinated calcium hydroxylapatite (Ca10(PO4)6F2*x*(OH)2–2*x*, where *x*

perturbations of fluorine environments, involving the displacements of both fluorine and hydroxyl groups from their normal positions. This leads to a distortion of the electronic environment with regard to the investigated fluorine nucleus and gives reasons for ob‐ served change in the 19F chemical shift tensor of fluoridated hydroxylapatite with different fluorine content. Furthermore, the presence of OH-group destroys the fluoride long-range structure and that results in an isotropic chemical shift distribution. This leads to observed

The scanning microscope (SEM) permits the observation and characterization of heterogene‐ ous organic and inorganic materials on a nanometer (nm) to micrometer (μm) scale. In SEM, the area to be examined or the volume to be analyzed is irradiated with finely focused electron beam, which may be swept in a raster across the surface of the specimen to form an imager or

parameters and the content of fluorine in apatite. The presence of OH<sup>−</sup>

increase in the 19F line width in the case of low fluorine content [104],[107].

**3.1.10. Scanning electron microscopy, structure and elemental analysis**

) indicates the correlation between 19F chemical shift tensor

groups induces

H, 19F and 31P NMR spectra

Secondary and Auger electrons are highly susceptible to elastic and inelastic scattering and can leave the specimen only from a very thin surface layer of the thickness of a few nanome‐ ters. The most probable energy of BSE falls into the broad part of the spectrum in **Fig. 8**(**b**), but they also show more or less pronounced elastic peak followed by plasnom losses, which depend on the primary energy, the take-off angle and the tilt of the specimen. Continuously slowing-down approximation assumes that the mean electron energy decreases smoothly with decreasing path length of the electron trajectories inside the specimen. The maximum information depth of BSE is of the order of half the electron range. Characteristic X-rays will only be excited in the volume in which the electron energy exceeds the ionization energy of the inner shell involved. Inelastic scattering in semiconductors results in the generation of electron-hole pairs. The recombination can take place without radiation but may result in the emission of light quanta (cathodoluminescence, CL) [111].

The method known as electron backscattering diffraction (EBDS) enables to determine the crystal structure and grain orientation of crystals on the surface of specimen. To collect maximum intensity in the diffraction pattern, the surface of specimen is stipple tilted at an angle of typically 70° from the horizontal (**Fig. 22**(**a**)). The intensity of backscatter **Kikuchi patterns** (please see the pattern of fluorapatite in **Fig. 23**) is rather low, as is the contrast of the signal, so extremely sensitive cameras and contrast enhancement facilities are required. This pattern allows to identify the phases and shows the misorientation across the grain bounda‐ ries [108].

Scanning electron microscope can be also used to determine compositional information using characteristic X-ray. The development of instruments for obtaining localized chemical analysis of solid samples, i.e. electron probe microanalyzer (EMPA), occurred at the same time as the development of SEM.

Scanning electron microscopy (SEM) is used for grain interactions and spot analysis [98],[112], electron microprobe microanalysis (EPMA) for the distribution of elements in the matrix, investigation of the effects of impurities on the properties of apatites and investigation of reaction interface [113],[114],[115],[116].
