## 4 Results
### Binding Media by Means of GC-MS  
In GC-MS investigations, the procedure consisted of two main steps: the first one focusing on the lipidic components and the second on the proteinaceous material. Two chromatograms were collected for each sample: the first one from fatty acid derivatives and the second from amino acid derivatives. The GC-MS analysis carried out on the samples of _Visitation_ altarpiece (SF1, SF2, SF4), _Martvydrom of Saint Sebastian_ panel (GSS7, GSS8) and _[PERSON] and the [PERSON]_ canvas (GSM12, GSM13) showed the presence of fatty acids and amino acids. Azelaic (nonanediolic acid, saturated dicarboxylic acid), muristic (C14:0), palmitic (C16:0), oleic (C18:1) and stearic (C18:0) acids were detected (Table S5). In our samples, the value of the azelaic acid/palmitic acid ratio (A/P) is lower than 1 (Table S5) and according to the literature, this value would lead to excluding the presence of any siccative oil [52]. In an oil and egg mixture, the high palmitic acid content of egg yolk can probably alter the A/P (and also palmitic/stearic) ratio. Furthermore, some scholars mentioned the absence of dicarboxylic acids in oil paintings, as in [PERSON]'s samples, probably due to their low methylation recovery in the transesterification reaction [53]. Azelaic acid  
Figure 5: _Martvydrom of Saint Sebastian_ panel. FTIR-ATR spectrum of the (**a**) pinkish and (**b**) red pictorial layers of sample GSM6.  
is a characteristic marker of aged drying oils produced during the oxidative degradation of fatty acids and the A/P ratio is not constant with time [54; 55]. Moreover, the presence of inorganic pigments or organic lacquer could affect the drying process and, as a consequence, the fatty acids profile [56]. [PERSON] indicated that a low ratio (0.52 and 0.80) between azelaic and palmitic acid (A/P) is the result of the drying process of the lipidic fraction [57].  
As the ratio between palmitic and stearic acid (P/S) remains stable over time, it can be considered a useful parameter to obtain information about the type of drying oil used by the artist, i.e., linesed, walnut or poppy seed oils. In fact, values of P/S in the range 1.4-2.1 could refer to linesed oil; 2.4-2.9 to walnut oil and 2.9-4.1 to poppy seed oil [52]. In all the samples, P/S showed values in the range of 1.59-1.75. According to the sub-mentioned statement and being aware that [PERSON]'s artworks were subjected to various restoration interventions during the centuries, especially the _Martyrdom of Saint Sebastian_ panel and the _Tobias_ and _Archangel Raphael_ canvas [5], it was assumed that the low value of azelaic acid may be due both to its partial removal during the conservative history of the paintings and to the repaint intervention containing fresh oil with a low content of azelaic acid. For this reason and due to the results of FTIR analysis, the presence of linesed oil was, however, considered.  
The chromatograms related to the proteinaceous material were considered to obtain the quantitative evaluation of the amino acidic profile, obtained after hydrolysis. The GC-MS investigation showed the presence of amino acids in all the samples. The data obtained were evaluated and subjected to PCA. In the PCA score plot (Figure S9), the samples from the _Visitation_ altarpiece are aggregated in the positive PC1 quadrant related to the egg standard area. The samples from _Martyrdom of St. Sebastian_ (GSS7, GSS8) fitted with animal glue and casein (skimmed milk) standards while the samples from _Tobias and the Archangel Raphael_ (GSM12, GSM13) are in the area of the casein reference data [25].