## 3 Results and Discussion
### Analysis of Moratars by Means of SEM/EDS
#### 3.3.2 GC/MS Analyses  
The investigations by means of GC/MS identified lipid and proteinaceous materials in the samples investigated (BB3, BB4, BB5 and BB6). The lipid fraction detected is not attributable to a drying oil, suggesting that the oil technique was not used in this painting. We found azelaic (nonanediolic acid, saturated dicarboxylic acid), m  
Figure 4: FTIR spectrum of BB6 sample: c—calcite; g—gypsum; q—quartz; b—ultramarine blue; p—proteinaceous material.  
(C16:0), oleic (C18:1), and stearic (C18:0) acids, but the azelaic acid/palmitic acid ratio being lower than one excludes the presence of siccative oils [55; 56].  
Figure 5 shows the chromatographic profile of the BB3 sample.  
To identify the binding media, the amino acid percentage content in each sample was determined by comparison with the _Opificio delle Pietre Dure_ reference collection in Florence, Italy [57]: a dataset of 43 reference samples of egg (whole, egg white, egg yolk), casein, animal glue, and their mixtures (painted models simulating ancient polychromies). Principal component analysis (PCA) was carried out on the correlation matrix of the relative percentage content of eight amino acids (aspartic acid, glutamic acid, proline, hydroxproline, phenylalanine, alanine, glycine, and leucine) [58; 59; 17].  
The PCA score plot of the relative percentage content of the eight amino acids in the 43 reference samples and in the artistic samples, BB3, BB4, BB5, and BB6, is reported in Figure 6.  
It should be noted that the first main component segregates the reference samples into two large groups: the first (points on the right side of the Cartesian graph) includes those associated with egg (E), milk (M), and egg-milk (EM) and is characterized by the amino acid composition of leucine, aspartic acid, glutamic acid, and phenylalanine; the second group (points on the left of the graph), associated with glue (G), glue-egg (GE), and glue-milk (GM), is distinguished by the composition of the reference samples of alanine, glycine, hydroxproline, and proline. Moreover, in the first group, the second main component differs from the references (egg, upper box), characterized by the content in Asp and Leu, and the milk (lower box), typified by glutamic acid. In the second group, glue-egg (typed by glycine) is diversified--but to a lesser extent--all falling in the upper box, from glue-milk (with a less typical amino acid content); most of them fall into the lower box.  
Figure 5: GC/MS chromatogram of the proteinaceous fraction of BB3 sample (dark-green sample taken from cypresses, exfoliated brushstroke, west wall). Ala—alanine; Gly—glicine;Thr—threonine; Ser—serine; Val—— valine; Nval—normalize (internal standard); Leu—leucine; Nieu—norleucine (internal standard); Pro—proline; Hyp—hydroxyproline; Asp—aspartic acid; Glu—glutamic acid; Phe—phenylalanine.  
The samples collected were placed on the Cartesian diagram of the PCA not far from the glue-egg references, differing in their peculiar content of Alanine, suggesting a combination of egg and animal glue binders in all the samples.  
The same mixture of binders found in samples BB3, BB4, BB5, and BB6 (Table 3) suggests the use of the \"_a secco_\" technique also incorporating pigments that usually are employed in \"_a fresco_\" technique. The lipid fraction, mainly palmitic, oleic, and stearic acids, found in the samples, is probably due to egg yolk lipid content.