*10.3.1 Differential scanning calorimetry (DSC) and thermal denaturation temperature (Td)*

Any DSC calorimeter brand can be used, such as a Perkin Elmer DSC7. The thermal behaviour; stability of the native molecular structure and denaturation of collagen can be determined by carrying out differential scanning calorimetry (DSC). Denaturation temperature is obtained from the transition in the baseline in the 30–80°C region by taking the inflexion point reading. Total denaturation enthalpy (ΔH) can be estimated by measuring the area in the DSC thermogram.

Collagen denaturation temperature (Td) depends on collagen water content, collagen extraction method, collagen source, degree of collagen cross-linking and hydroxyproline content. Thermal stability of the collagen triple helix depends on hydrogen bonds (inter- and intra-hydrogen bonding) which further influences the folding and unfolding process when hydrogen bonds are broken and connected [134, 135]. Hence, the thermal stability of collagen depends on the cross-linking of collagen molecules (inter and intra).

Due to the polymeric nature of collagen, the thermal-induced denaturation of collagen is usually complicated. Heating collagen in wet or dry state reveals a series of thermal transitions. Thermal denaturation of collagen occurs due to hydrogen bonds breaking and hence the unfolding of the triple helices forming random polypeptide coils [136]. Cross-linking among the collagen molecules increase and mature with age and provides further stability. The age-related accumulation of cross-links increases the thermodynamic stability of collagen by increasing the activation energy required for collagen denaturation. However, the maturity of collagen cross-linking is limited to the functionality of the tissue. Post-mortem cross-linking of collagen can increase to the point where the tissue may become brittle [137].

Within the collagen fibril, there are complex interactions within and between the packed molecules. In addition to inter, intramolecular cross-links, and different forms of cross-linkages, there are several additional hydrophobic and ionic interactions that must be accounted for regarding collagen denaturation. The presence of non-collagenous components in the extracted collagen sample can cause variations in thermal denaturation [138].

Due to the domain structure of the triple helix, not all parts of the collagen molecule may denature at the same rate and it is almost impossible to define a definite equilibrium Td. Studies have also shown an increase in Td with an increase in hydroxyproline content [61, 139].

#### *10.3.2 Thermogravimetric analysis (TGA)*

Thermal stability of extracted collagen is investigated using a gravimetric analyser. Approximately 5–10 mg of the sample can be used. The mass loss is

## *Collagen: From Waste to Gold DOI: http://dx.doi.org/10.5772/intechopen.94266*

recorded while the sample is heated from room temperature up to 800°C at a rate of 10°C per minute. The first derivative of percentage mass change versus temperature can also be calculated to investigate temperature regions where mass loss was occurring.

Ramanathan et al. [140] used TGA to assess the thermal stability of fish skin collagen which was extracted via acid-solubilisation. They report using samples of approximately 5 mg and heating samples at 10°C/min in the temperature range of 0–800°C. The acid-solubilised collagen showed two weight loss steps on the TGA thermogram, relating the first stage to the loss of structural and bound water and stage two to thermal degradation of the polypeptide chain. The study concluded to show that the two peaks observed on the TGA differential curve were of collagen denaturation and collagen degradation respectively.
