## 3 Results and Discussion
### Organic Binding Media  
The study, which aimed to identify the organic binders, began by analyzing the blue (B1), yellow (B19 and B20), gilding area (B2) and green (B7) collected samples, by means of FTIR spectroscopy. Typical bands of proteinaceous material--i.e., the characteristic CH stretching (2924 and 2954 cm\({}^{-1}\)), C=O stretching of amide I (1643 cm\({}^{-1}\)) and N-H bending of amide II (1539 cm\({}^{-1}\))--were detected. Based on these results, GC-MS was used to identify the lipidic and proteinaceous materials.  
Only the gilding sample B2 gave both lipidic and proteinaceous content. The analysis of the lipidic fraction, performed by GC-MS, revealed azelaic acid (CC9), palmitic acid (C16:0) and stearic acid (C18:0) as the main fatty acids (Figure 9).  
Figure 7: Raman spectra of white pigments. H = hydrocerussite.  
The presence of azelaic acid, and the absence of polyunsaturated fatty acids, are characteristic of an oxidized lipidic medium, being dicarboxylic acids the oxidation products of polyunsaturated acyl chains during the aging processes [21,22].  
Figure 8: Raman spectra of different oxides found in the gilding material, and used for restorations.  
Figure 9: GC-MS chromatogram of the lipidic fraction of the B2 sample (gilding sample with fragments of gold taken from the central garland around the Christ). CC9 = azelaic acid, C16:0 = palmitic acid, C17: tetracosane (internal standard), C18:0 = stearic acid.  
The value of the azelaic/palmitic acid ratio indicated the presence of drying oils in the sample.  
The palmitic/stearic acid (P/S) ratio is stable over time, and can give information about the type of drying oil used by the artist, i.e., linesed, walnut or poppy seed oils [23; 24]. Values of P/S in the ranges 1.4-2.1, 2.4-2.9 and 2.9-4.1 were obtained indicating linseed oil, walnut oil and poppy seed oil, respectively [10]. The P/S value 1.4 observed for the gliding B2 sample, suggested the presence of linseed oil. Our hypothesis is that Au foil had been applied on a brown background where the Au foil is applied on the oil-based mission [17]. In the 'a missione' technique, drying oils (typically linseed oil), were used to glue the metal foils [24]. The oil gliding technique appears to have been described by [PERSON], and widely used in painting between the 14 th and 15 th centuries [25].  
Chromatograms of samples B1, B2, B7, B19 and B20 show the amino acids features. Figure 10 shows the chromatographic profile of B7 sample.  
To identify the binding media, the percentage content of amino acids in each sample was compared to those from a dataset of 43 reference samples of egg (whole, egg white, egg yolk), casein and animal glue, belonging to the reference collection of the Opificio delle Pietre Dure in Florence, Italy [26]. Principal component analysis (PCA) was done on the correlation matrix of the relative percentage contents of eight amino acids (asparatic acid, glutamic acid, proline, hydroxyproline, phenylalanine, alanine, glycine and leucine) [27; 28].  
The PCA score plot is described in Figure 11: it locates all the samples in a new cluster suggesting a combination of egg and animal glue binders in all the samples.  
The presence of the same mixture of binders in samples B1, B7, B19 and B20, where Raman analysis evidenced lazurite, malt, hematite, green earth and goethite, allowed us to hypothesize the use of the a secco technique also with pigments that did not require the employment of organic media.  
The B2 sample, being a gliding, shows the use of linseed oil, and of egg and animal glue.  
Figure 10: GC-MS chromatogram of the proteinaceous fraction of B7 sample (green sample taken from the meadow under the horse of St. Paul). Ala = alanine, Gly = glicine, Thr = threonine, Ser = serine, Val = valine, Nval = norvaline (internal standard), Leu = leucine, Nleu = norleucine (internal standard), Pro = proline, Hyp = hydroxyproline, Asp = aspartic acid, Glu = glutamic acid, Phe = phenylalanine.