Chemical Structural Analysis of Bone by Spectroscopy - Vibrational Spectroscopy for Tissue Analysis - page 192

was immersed in 5 mL of 99% anhydrous hydrazine in a glass-stoppered
flask and treated as follows: at room temperature for 1 h; hydrazine reagent
changed; incubated at 60°C for 1 h; hydrazine reagent changed; incubated
at 60°C for 15 h; hydrazine reagent changed; and incubated at 60°C for 24 h.
The bone samples were then immersed in a graded series of aqueous alco-
hol solutions (5 mL, 30 min each) of 50, 75, 87.5, 100, 100, and 100% concen-
trations. The samples were then immersed in 5 mL of 95% acetone for 2 h.
Finally, the samples were placed in a desiccator and left to dry at room tem-
perature for 48 h before analysis was carried out.
raman Spectral Data acquisition
Raman spectra of the bones and hydroxyapatite samples were recorded using
a Nicolet spectrophotometer, equipped with a Nd:YVO 4 near-infrared laser,
which eliminated the problems of sample fluorescence and photodecompo-
sition. The low-power near-infrared laser in the Raman spectrophotometer
makes it possible to analyse fresh tissues without loss of sensitivity due to
the fluorescence of organics. The analysed bone samples were attached to
a metal post (sample holder). Hydroxyapatite, calcium carbonate, calcium
phosphate, calcium hydroxide powder, and sodium hydroxide pellet sam-
ples were analysed in a glass tube 5 mm in diameter, similar to that used
in nuclear magnetic resonance (NMR) spectroscopic analysis. Scattered
radiation was collected and collimated by 180 o reflective optical geometry.
The spectra were obtained by using 450 mW of laser power, 100-600 scans
(depending on the samples), and 4 cm −1 resolution.
Spectral Findings
The most striking feature of the spectra of bone tissues, (without deproteination)
obtained from this work was the absence of fluorescence from the samples
(Figures 7.1 and 7.2), allowing well-defined peaks to be resolved. Raman spec-
troscopy of the whole bone samples revealed numerous peaks associated
with an organic phase, i.e., the peaks in the 3000-2800 cm −1 region were due to
C=H and the peaks at 1650-1600 cm −1 were due to the amide band of collagen
(a combination of C=O stretching, C=N stretching, and N=H bending modes).
The human, bovine, baboon, and sheep tissue provided almost identical
spectra, both in the fresh and deproteinated conditions (Figures 7.1 and 7.2
and Table 7.1). The deproteination procedure clearly resulted in a consider-
able decrease in the scattering intensity of peaks arising from the organic
phase. The peaks in the region of 3000-2900 cm −1 wavenumber were due to
the C-H stretching mode and 1450 cm −1 to the C-H, bending mode, while
other peaks, which were present due to the inorganic phase of the bone,
remained substantially unaffected.
The peaks for the phosphate moiety centred at 952 cm −1 (PO:- stretching
mode) and 584 cm −1 (PO:- bending mode) remained unaffected. Similarly,