# Effects of Temperature on the FT NIR Raman Spectra of Fish Skin Collagen
## 3 Results and Discussion
### Temperature Effect on the FS Collagen  
Temperature studies of Raman spectra of FS and BAT collagen were carried out in the temperature range from 303 K to 403 K. In Figure 3a,b, temperature changes of FS collagen and BAT collagen Raman spectra are presented, respectively. Raman spectra of FS collagen do not change up to T \(\cong\) 353 K, and above 353 K Raman spectra undergo irreversible change. The most significant changes are observed for the lines at 860, \(\sim\)918, 935, 1171 cm\({}^{-1}\) and for the bands related to amide I and amide III vibrations. The sharp line at 860 cm\({}^{-1}\) in the spectrum at 303 K, assigned to tyrosine, is significantly less intense at 403 K (Figure 4a). The doublet of lines at 918 and 935 cm\({}^{-1}\), assigned to \(\alpha\)-helix conformation, shows a considerable decrease in their intensities at 403 K. The most significant changes in high temperature are observed for the bands related to both amide I and amide III vibrations. A substantial shift of the band at 1274 cm\({}^{-1}\) to \(\sim\)1270 cm\({}^{-1}\) (Figure 5a) assigned to the \(\alpha\)-helix may indicate its considerable decrease and an increase in the content of both \(\beta\)-sheets and the disordered part of the polypeptide chain. On the other hand, in the part of the Raman spectrum related to the vibrations of amide I (Figure 5c), one can observe a shift of a narrow band centered at 1666 cm\({}^{-1}\) to a higher frequency at \(\sim\)1670 cm\({}^{-1}\). Moreover, a decreasing intensity of the line suggests a reduction in the content of \(\alpha\)-helix conformation and an increase in the content of the \(\beta\)-sheet phase. We can also observe a significant widening of Raman bands, indicating increased structural disorder (Figure 5b). Figure 4: Comparison of Raman spectra of FS collagen measured at 303 K and 403 K in the frequency ranges 800–1200 cm\({}^{-1}\) (a) and 1200–1750 cm\({}^{-1}\) (b). Figure 3: Temperature changes of Raman spectra for FS collagen (**a**) and BAT collagen (**b**) in the frequency range of amide III and amide I vibrations. To conclude, the temperature studies of the Raman spectra of FS collagen revealed conspicuous changes occurring in the \(\sim\)358 K to \(\sim\)363 K temperature range. Heating of FS collagen to 403 K causes irreversible changes in its secondary structure. Comparing the Raman spectra of BAT and FS collagen measured at 403 K (Figure 6), one can see their striking similarity, whereas at room temperature considerable differences can be observed (see Figure 2). Temperature studies of Raman spectra changes in BAT collagen showed that the character of Raman spectra does not undergo any change in the temperature range from 300 K to 403 K (Figures 3b and 7). Additionally, the Raman band positions change linearly with increasing temperature (Figure 8), not exhibiting any anomalous behavior. Figure 5: Temperature dependence of the Raman band position of FS related to amide III vibration (**a**) and its spectral width (**b**), amide I vibration (**c**) and its spectral width (**d**). The lines are guides for the eye. Figure 6: Comparison of Raman spectra of FS and BAT collagen in the range of amide I and amide III vibrations measured at 403 K.  
Analyzing the thermal changes in the amide groups for both FS and BAT types of collagen, we compare the Raman intensity ratios of the most intense bands at \(\sim\)1270 cm\({}^{-1}\) (precisely \(\sim\)1275 cm\({}^{-1}\) for FS and \(\sim\)1270 cm\({}^{-1}\) for BAT) for amide III and \(\sim\)1660 cm\({}^{-1}\) (1666 cm\({}^{-1}\) for FS and \(\sim\)1656 cm\({}^{-1}\) for BAT) for amide I with the intensity of the band at \(\sim\)1450 cm\({}^{-1}\). The band at \(\sim\)1450 cm\({}^{-1}\) is related to the CH\({}_{2}\) bending vibration, which is practically insensitive to the secondary structure. Table 2 presents the Raman band intensity ratios at 303 K and 403 K for both types of collagen. Figure 8: Temperature dependence of the Raman band position of BAT collagen related to amide I vibration. The line is a guide for the eye.