*2.7. Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC)*

In order to confirm the purity, the extracted blue shark skin collagens were eluted through RP-HPLC column and compared with standard bovine collagen. As shown in Figure 4, there were two main chromatogram peaks eluted at 3.427 and 19.0 min for ASC and 3.407 and 22.980 min for PSC, respectively. The first peak observed at 3.4 was the acetic acid peak. The present observation was in accordance with a previous report [24]. Compared to the standard bovine collagen, the blue shark fish collagen showed a dissimilar chromatographic pattern. We speculated that this might be due to the difference in their molecular arrangements between mammalian and fish collagen. Also, the RP-HPLC pattern confirmed that extracted collagen was pure and free from non-collagen protein and other contaminants, further justifying the SDS-PAGE data of collagen (Figure 2A).

**Figure 4.** RP-HPLC elution profile of blue shark skin collagen. ASC-Acid soluble collagen, PSC-Pepsin soluble collagen.

#### *2.8. Thermal Properties*

Thermal properties such as the material weight loss towards temperature and the thermal disintegration configuration of the blue shark skin collagens were evaluated by the thermogravimetric analyzer (TGA). From the TGA curves, 50% weight loss in ASC and PSC were observed at 339 and 342 ◦C, respectively (Figure 5A). At high temperature (650 ◦C), the mass weight of ASC and PSC were about 22.53 and 23.64, respectively.

**Figure 5.** Thermogravimetric analysis (TGA) (**A**) and differential scanning calorimetry (DSC) (**B**) of blue shark skin collagen. ASC-Acid soluble collagen, PSC-pepsin soluble collagen.

ASC and PSC from blue shark skin had two endothermic peaks, which denoted the thermal denaturation and melting temperature. The denaturation temperature of ASC and PSC was about 28.3 and 29.8 ◦C, respectively (Figure 5B). The degree of hydroxylation and certain specific amino acid residues such as Gly-Pro-Hyp might determine the denaturation temperature of collagen [15]. It was observed that imino acids play a major role in the thermal properties of collagen. For instance, the hydroxyproline mainly contributes to the stability of the helix chain [25]. In this study, PSC had better thermal properties than ASC due to the high content of iminoacids.
