## 4 Discussion
### Artificial Ageing Protocol (AAP)  
#### 4.1.1 Colorimetric Analysis  
With regard to the color, after ageing, in the case of the original polychrome bands (Band A), it is possible to establish with respect to the pigments used, azurite and vermilion, that both are altered by the ageing process, especially when they are bound with animal glue [36]. In the case of azurite, the chromatic alteration occurs when agglutinated with animal glue and is due to a loss of clarity (\(L^{*}\)(D65)), indicative of a discoloration of the pigment, due especially to the relative humidity applied during the ageing process. In the case of the vermilion pigment, there is a loss of clarity, and especially chroma (loss of red), with both binders; this is due to the combination of UV light and relative humidity applied during the ageing process, as both parameters generate a process of photodegradation and oxidation in this pigment [48; 49]. In the case of the band referring to the linseed oil repainting, it is possible to appreciate that the ochre pigment, due to its composition FeO(OH) + Al\({}_{2}\)O\({}_{3}\) + SiO\({}_{4}\) + CaCO\({}_{3}\)), is more stable to the ageing process than the mixture of artificial ultramarine blue pigment (Na\({}_{\text{8-10}}\)Al\({}_{6}\)Si\({}_{6}\)O\({}_{29}\)S\({}_{2}\)-4) with titanium white. This is because the ultramarine pigment can be affected by the acidity of the binder used [49]. 1.2 Fourier Transform Infrared Spectroscopy (FTIR) and Gas Chromatography Coupled with Mass Spectrometry (GC-MS)  
The study of chemical alterations through Fourier Transform Infrared Spectroscopy (FTIR) and Gas Chromatography coupled with Mass Spectrometry (GC-MS) shows that, in the case of the pigments used (azurite, vermilion, ochre, and the mixture of ultramarine blue and titanium white), they have not been chemically affected by the ageing process. As previously stated, the change experienced is chromatic and is directly related to the binders used. Therefore, it is necessary to analyze what potential chemical changes these organic components may have undergone. In the case of the animal glue binder, the results obtained through Fourier Transform Infrared Spectroscopy (FTIR) revealed a marked decrease in the absorbance of the amide II band in the aged sample containing vermilion and animal glue (P2 vCa(A) aged). This suggests partial denaturation or degradation of the proteinaceous components and, therefore, a chemical alteration. In comparison, the aged sample with azurite and animal glue (P1 aCa(A) aged) exhibited greater chemical stability, although a reduction in both amide I and II bands was still observed. This indicates that, although the structural damage to the protein matrix was less severe, there was still an alteration in the binder's conformation. Such chemical structural changes are consistent with the expected effects of accelerated ageing, particularly under conditions of UV radiation and elevated temperatures, as commonly observed in animal glue binders [36; 37]. On the other hand, amino acid composition analyses using Gas Chromatography Mass Spectrometry (GC-MS) after artificial ageing revealed notable stability in the proportion of the main amino acids, glycine, proline, hydroxyproline, glutamine, and alanine, with an average preservation rate of 22% across all samples. This stability suggests that, despite the chemical changes indicated by Fourier Transform Infrared Spectroscopy (FTIR), no significant degradation of the amino acid chains comprising the animal glue occurred. The apparent contradiction between Fourier Transform Infrared Spectroscopy (FTIR) and Gas Chromatography coupled with Mass Spectrometry (GC-MS) data may be interpreted as surface-level degradation or conformational changes in the protein structure, rather than complete breakdown of the peptide chains. This hypothesis is supported by previous studies that demonstrated the relative stability of certain amino acids, such as alanine, glycine, leucine, and proline under ageing conditions [43; 50].