# Effects of Temperature on the FT NIR Raman Spectra of Fish Skin Collagen
## 3 Results and Discussion  
### Raman Spectra of FS and BAT Collagen Measured at Room Temperature  
The Raman spectra of FS and BAT collagen recorded at 303 K are presented in Figure 2.  
Table 1 presents the assignment of the Raman bands for FS and BAT type collagen at room temperature. To assign the Raman bands of samples used in this study, the results of vibrational studies of different samples containing different kinds of collagen as well as proline, hydroxyproline and glycine separately were utilized [35, 36, 37, 45, 51, 52, 53, 54, 55, 56, 57, 58, 59]. In the Raman spectra of collagen, one can distinguish four principal frequency ranges: the first at 200-600 cm\({}^{-1}\), assigned to the vibrations of the triple helix [44, 59], the second at 800-1200 cm\({}^{-1}\), assigned mainly to the vibration of proline and hydroxyproline as well as secondary structure of collagen, and the third at 1200-1750 cm\({}^{-1}\), where the bands related to amide I and amide III vibrations were observed. The fourth frequency range from 2800 to 3200 cm\({}^{-1}\) presents the band related to CH\({}_{2}\) group vibrations. \begin{table}
\begin{tabular}{l l l l} \hline \hline
**FS** & **BAT** & **Assignment** & **Assignment** \\
**Band Position/cm\({}^{-1}\)** & **Band Position/cm\({}^{-1}\)** & **Assignment** & **Assignment** \\ \hline
282 w & 292 ww & 3-helix \\ \hline
314 w & 307 vw & 3-helix \\ \hline
388 vw & 396 w & \\ \hline
535 w & 530 w & \(\delta\)(CCN) & \\ \hline
639 w & 644 vw & \(\delta\),\(\omega\)(COO\({}^{-}\)) & \\ \hline
649 w & & \\ \hline
760 w & 760 w & \(\
u\)(CCO) & \\ \hline
813 w & 815 w & \(\
u\)(CCO), \(\
u\)(CC) & \\ \hline
822 w & & \\ \hline
850 wsh & 854 m & \(\
u\)(CCO), \(\
u\)(CC) & Pro \\ \hline
861 m & & \\ \hline
870 wsh & 874 m & \(\
u\)(CC) & Hyp \\ \hline
918 m & 918 m & \(\
u\)(C-COO\({}^{-}\)) & \(\alpha\)-helix \\ \hline
935 m & 935 m & \(\
u\)(CC) & \(\alpha\)-helix \\ \hline
962 w & 964 w & \\ \hline
975 w & 974 & \\ \hline
1003 w & 1002 m & \(\
u\)(CC) aromatic ring & phenylalanine, tyrosine, tryptophan \\ \hline
1029 wsh & 1030 m & \(\
u\)(CCN) & phenylalanine \\ \hline
1045 w & 1042 wsh & & \(\beta\)-sheet \\ \hline
1060 sh & 1064 m & \(\omega\)(CH\({}_{2}\)) & \(\beta\)-sheet \\ \hline
1085 w & 1082 m & \(\
u\)(CN) & \\ \hline
1096 w & 1096 msh & \(\
u\)(CN) & \\ \hline
1124 & 1123 msh & & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Assignment of the Raman bands of FS and BAT types of collagen at room temperature. Figure 2: Raman spectra of FS and BAT types of collagen in the range from 250 to 1200 cm\({}^{-1}\) (**a**), in the range of amide I, amide III and the range of CH\({}_{2}\) vibrations (**b**). Raman spectra were recorded at 303 K.  
The Raman lines at 282 and 314 cm\({}^{-1}\) are clearly visible for FS collagen, whereas similar lines at \(\sim\)292 and 307 cm\({}^{-1}\) for BAT collagen are broadened and exhibit low intensities. Some authors [41; 53] assign the lines in the range 200 to 400 cm\({}^{-1}\) to the vibrations related to a triple helicity of the collagen. A significant difference between FS collagen and BAT collagen is observed in the frequency range 830-900 cm \({}^{-1}\). The doublet of lines at 854 and 874 cm\({}^{-1}\) for BAT collagen is assigned to \(\delta\)(CCH) aromatic and \(\
u\)(CC) proline and hydroxyproline vibrations, respectively. In the case of FS collagen, one intense band with weak shoulders is observed at 861 cm\({}^{-1}\). After deconvolution of the band, the shoulders occur at \(\sim\)854 cm\({}^{-1}\) and \(\sim\)870 cm\({}^{-1}\). The band centered at 861 cm\({}^{-1}\) may be assigned to the vibrations related to tyrosine. The shoulders with low intensities at \(\sim\)854 and \(\sim\)870 cm\({}^{-1}\) may be related to the vibrations of proline and hydroxyproline. The remaining Raman bands occurring in this frequency range appear in similar positions and are characterized by similar intensities, both for BAT and FS.