## 3 Materials and Methods
### Micro Destructive Analyses
#### 3.2.2 Methods  
As infrared spectrophotometer, an ALPHA (Bruker Optik GmbH) FTIR spectrometer equipped with a Globar IR source, an interferometer (Rock-Solid)tm, insensitive to external vibrations and able to work in any spatial orientation), a DLaTGS room temperature detector and an ATR accessory with a diamond crystal were used. The IR spectra were acquired in the painting surface and in the lacunas in the spectral range 4000-375 cm\({}^{-1}\) with a spectral resolution of 4 cm\({}^{-1}\) and 128 interferograms. GC\(-\)MS analysis was carried out on a Hewlett Packard HP 6890 gas chromatograph coupled to MSD--HP 5973 MSD mass selective detector (Hewlett-Packard) and a split-splitless injector were used. The mass spectrometer was operated in the EI positive mode (70 eV). The carrier gas was helium. The injector was kept at 280 \({}^{\circ}\)C. MS conditions were as follows: interface temperature, 280 \({}^{\circ}\)C; ion source temperature, ca. 230 \({}^{\circ}\)C; scan mode in the range of \(m/z\) 40-400. A low-polarity capillary column SLB-5 ms by Sigma-Aldrich Supelco (5% phenyl groups, I.D. 30 m \(\times\) 0.25 mm, film thickness of 0.25 \(\upmu\)m) was used. An analytical procedure for the characterization of polysaccharides and the identification of plant gums in reference materials and paint samples is defined. The procedure is based on hydrolysis with 2 N trifluoroacetic acid (120 min, 121 \({}^{\circ}\)C), keeping in shaking. After letting it cool, take 150 \(\upmu\)L and put it in a flask together with 80 \(\upmu\)L phenyl-\(\beta\)-glucoside 1000 ppm (internal standard), and dry to rotary evaporator. Next, the residual acid is removed by adding 1 mL of methanol to the flask, which is then evaporated again with the rotary evaporator. Since uronic acids (such as glucuronic and galacturonic acid) that may be present in the sample are degraded by the trifluoroacetic acid which transforms them into lactones, 1 mL of 0.5 M ammonia dissolved in water is added. In this way, by bringing the pH to alkaline values, the reopening of the lactone ring and the return to the initial structure of the uronic acids are allowed, which are thus detectable by subsequent instrumental analysis. Ammonia is allowed to react for about 10 min, then evaporates again to the rotary evaporator. Finally, 800 \(\upmu\)L of Dimethylformamide and 200 \(\upmu\)L of N, O-Bis(trimethylsilyl) trifluoroacetamide (BSTFA) are added. It is allowed to react for 1 h at 60 \({}^{\circ}\)C under stirring and injected into GC-MS. The samples MY32 and MY46 were also treated with the basic methodology for the identification of fatty acids and amino acids already carried out in [PERSON] et al, 2012 [25]. Raman spectra were collected on cross-section samples using an Olympus BXFM Microscope coupled with a LabRam HR800 spectrometer ([PERSON], France) fitted with an air-cooled CCD detector (1024-256 pixels), set at \(-\)70 \({}^{\circ}\)C. The spectrometer had a focal length of 80 mm and the spectra were by a 600 grooves/mm grating, a 100 \(\times\) objective and a He-Ne laser (632.8 nm) as excitation source with the resolution of 4 cm\({}^{-1}\). The acquisitions were achieved with the laser powers between 0.2 and 4 mW and the exposure time varied between 5 and 10 s with 10 accumulations. The spectrometer was calibrated and checked with silicon at 520 cm\({}^{-1}\). Microscope observations were performed on micro-samples using an OLYMPUS BX51 polarized light microscope in reflected and transmitted light mode using reflected polarized and UV light. The microscope was directly connected to an Olympus SC50 camera equipped with Stream Basic software for images acquisition. All the wall paintings' samples (Table 1) were embedded in an epoxy resin and carefully polished after curing of the resin with progressively finer paper to be made into cross-sections. Some samples were carefully ground to final thickness (usually 30 \(\upmu\)m) to be made into thin sections too.