2.6. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC)

Thin membrane samples (10 × 10 mm, 1.5 mm thick) 12–1.5, 24–1.5, 48–1.5, and 96–1.5 were prepared as described above. Additional samples for 12–2 and 96–1 groups were prepared, representative of low and high ED, respectively. All samples were subjected to our standard rinsing protocol in PBS and then lyophilized. The 12–2 and 96–1 samples, and uncrosslinked lyophilized keratin, were subjected to thermogravimetric analysis (TGA, TA Instruments, New Castle, DE - Energy Research Center, University of Maryland) heating the samples up to 350 °C at 10 °C/min under 100% N₂. TGA was performed to identify variations between crosslinked and uncrosslinked samples, and to determine the temperature ranges where these occurred. The 1.5 mm thick samples, and uncrosslinked keratin controls, were massed using a micro-balance (Sartorius ME-5, Gottingen, Germany) and sealed into differential scanning calorimetry (DSC) pans (10–15 mg per pan). DSC was performed on a Q100 differential scanning calorimeter (TA Instruments, New Castle, DE) by heating the samples from 30 to 300 °C at 10 °C/min under 100% N₂. Resulting thermograms were compared to those of uncrosslinked controls to determine the crosslinking degree (CD) as previously reported in literature [34] (n = 9–15). Briefly, in DSC thermograms the area under the peak represents the maximum enthalpy change possible in the system. Changes in these areas are indicative of changes in the internal energy of the network and are used to quantify bond formation and CD. As such, enthalpy change was used to calculate CD [34], as:

where, ΔH (S0) is the reaction enthalpy of uncrosslinked reference (area under reference peak) and ΔH(Sχ)is the reaction enthalpy of the crosslinked sample (area under sample peak).