## 3 Results
### Detailed Analysis of FTIR Crystallinity Estimator at Different Polarisation Angles  
Pol-ATR-FTIR experiments were performed to obtain a better understanding of FTIR crystallinity estimators. Pol-ATR-FTIR spectra of a few fibroin fibers obtained with the linearly polarised light at different angles from 0\({}^{\circ}\) (A\(\parallel\), parallel spectra) to 90\({}^{\circ}\) (perpendicular spectrum, A\(\perp\)) are depicted in Figure 5. Amide I band was particularly interesting in these spectra, composed of parallel and perpendicular dichroism due to coupled modes in beta-sheets. In this vibration region, coupling between four peptide bonds is expected to result in four normal modes, out of which two are active and strong enough to be observed on IR spectra: v\(\parallel\)(0, \(\pi\)) and v\(\perp\)(\(\pi\), 0) [31]. Centered at 1699 cm\({}^{-1}\) v\(\parallel\)(0, \(\pi\)) band reflects the situation where two adjacent amide groups in the same chain vibrations are in-phase but are out-of-phase with adjacent chains amide groups (in antiparallel pleats), whereas centered at 1620 cm\({}^{-1}\) v\(\perp\)(\(\pi\), 0) band corresponds to the reversed situation where within the same chain amide groups vibe out-of-phase but in-phase with neighboring chains groups (in parallel pleats) [33]. Superimposed over these bands is one cantered at 1656 cm\({}^{-1}\) that describes oscillations of amide groups in \(\alpha\)-helix or random coil parts of spectra. Thus pol-ATR-FTIR spectra, recorded using a manually operated polarizer, show that changing polarisation states of light results in different absorption coefficients for MS sample absorption bands at distinct wavelengths (see Figure 4). Thus, the Amide I band is most intensive at 0\({}^{\circ}\) and Amide II at 90\({}^{\circ}\) with intermediate states at remaining angles. Figure 4: (**A**) ATR-FTIR spectra and normalized fibroin (**B**) Hydrolysis, (**C**) Oxidation and (**D**) Crystallinity estimators calculated from ATR FT-IR for model silk sample (MS) and historical: Inscription Banner (IB); Court Banner (CB); [PERSON]’s Funeral Banner (FB). Bands from the Amide I region were carefully analyzed, and the dichroic ratio \(R\) and order parameter \(S\) was calculated for sub-bands centered at 1699, 1956, and 1620 cm\({}^{-1}\). A significant degree of dichroism is observed for the bands referenced to \(\beta\)-sheet ordered regions (1699 and 1620 cm\({}^{-1}\)). \(R\) values around 3.322 and 0.352 (for 1699 and 1620 cm\({}^{-1}\), respectively) suggest a high degree of orientation to the fiber axis of IR activated transition dipole moment of the given vibration band (see Table 2). Crystalline regions vibrations show some ordered structure with values of orientation parameter \(S\) at 0.427 and \(-\)0.277, confirming in-phase and out-of-phase vibration corresponding to 1699 cm\({}^{-1}\) as parallel and 1620 cm\({}^{-1}\) as perpendicular to the crystalline domain axis [37]. Little dichroism is attributed to 1656 cm\({}^{-1}\), with the \(R\)-value close to 1 and \(S\)-value close to 0, as this is a band associated mainly with stretching vibration of C=O bond located in random coil and \(\alpha\)-helix domains [33]. According to the dichroic ratio \(R\) and order parameter \(S\)-values presented in Table 3 and Figure 6, the highly ordered orientation of silk vibrations in the crystalline domain changes artificial and natural aging conditions. \begin{table}
\begin{tabular}{c c c} \hline  & \(R\) & \(S\) \\ \hline
1620 cm\({}^{-1}\) & 0.352 \(\pm\) 0.010 & \(-\)0.277 \(\pm\) 9.67 \(\times\) 10\({}^{-5}\) \\
1656 cm\({}^{-1}\) & 0.718 \(\pm\) 0.016 & \(-\)0.104 \(\pm\) 2.43 \(\times\) 10\({}^{-4}\) \\
1699 cm\({}^{-1}\) & 3.322 \(\pm\) 0.122 & 0.427 \(\pm\) 1.49 \(\times\) 10\({}^{-2}\) \\ \hline \end{tabular}
\end{table} 
Table 2: Values of parameters \(R\) and \(S\) calculated for three different wavelengths from the collection of pol-ATR-FTIR for the initial model silk sample (MS).