## 3 Results and Discussion  
### Original Binding Media, Aged Coatings and Consolidants  
The presence of residues of synthetic origin (see following paragraph) caused an interference in the analysis of amino acids in sample SG_34; therefore, it was not possible to establish whether or not proteinaceous material was present. For all other samples, GC-MS analyses evidenced the presence of low amounts of proteinaceous material, which resulted above the limit of detection (LOD, 0.3 \(\upmu\)g of the eleven amino acids quantified).  
In sample SG_43, amino acids were present above the quantification limit (LOQ) of the analytical technique (0.6 \(\upmu\)g in total). The relative amino acid content was thus subjected to multivariate statistical analysis according to the principal component method (PCA) together with a database of reference samples containing animal glue, egg and casein [15]. The resulting score plot is shown in Figure 4, which indicates that sample 43 contains egg. This result is of considerable importance because the sample had been taken from an area of the top part of the vault, selectively scraping the pictorial surface: we believe egg is an original binder used by the artist. The remaining samples present amino acids below the LOQ, and it is therefore not possible to identify the proteinaceous binder. Animal glue is absent, though, thus ruling out the presence of animal glue [21; 22].  
Beeswax is identified by the presence of relatively high amounts of palmitic acid and tetracosanoic acid and (\(\upmu\)-1) hydroxy even-numbered fatty acids (with the most abundant 15 hydroxyhexadecanoic acid) (Figure 5), long-chain linear alcohols with a number of even carbon atoms (Figure S1), \(\upalpha\)-(\(\upmu\)-1)diols and linear aliphatic hydrocarbons with an odd number of carbon atoms (Figure 6) [7; 23]. On these bases, beeswax was clearly detected in all samples, with the exception of sample SG_44, and was particularly abundant in samples SG_37c and SG_43. Waxes were used as material for the consolidation and protection of wall paintings, as reported in many technical manual. This is the case in [PERSON]'s technical manual dated 1866, where restorers were advised to use mineral materials, such as paraffin, for the consolidation of wall paintings and painted facades [24; 25; 26]. The use of  
Therefore, the use of paraffin and mineral waxes is also plausible in the case of [PERSON]'s wall paintings.  
Beeswax makes the detection of glycerolipidic material difficult, as it substantially alters its chromatographic profile. The presence of relatively high amounts of azelaic acid, an oxidation product of polyunsaturated acids typical of glycerolipids of vegetable origin, points to the presence of an oxidized drying oil [29]. Traces were detected in both samples SG_30 and SG_44, but the chromatographic profile does not allow the determination of its nature, while relatively higher amounts were detected in samples SG_37c and SG_43 (SG_43 being a sample collected from the relief base for the application of gold leaf from the vault).  
The analysis of the lipid-resinous fraction also evidenced the presence of a Pinaceae resin given the identification in the chromatogram of trimethylsilyl esters of dehydro-abietic acid and 7-oxo-dehydrodiabetic acid [21]. These are clearly visible in the chromatogram of the SG_43 sample (Figure S2), but detectable in the chromatograms of all the samples, except for sample SG_41C, due to an analytical interference which compromised the silylation reaction.