## 3 Results
### The Trinity: A Seco Wall Painting
The protein-based nature of the binder is suggested by detection of amide infrared bands at ca. 3313 (amide A), 3064 (amide B), 1658 (amide I) and 1565 (amide II) cm\({}^{-1}\)[19]. Although, it is not possible to identify the type of protein exclusively by non-invasive FT-IR [20], a tentative assignment to an animal glue can be formulated considering the signals at ca. 4374 and 4260 cm\({}^{-1}\) of the methylenic _stretching_ and _bending_ combination bands [21] (Figure 3d). However, the broad shape of the bands and the co-presence of other compounds makes the protein discrimination difficult. Gypsum and calcium carbonate present in either the plaster, preparatory layers or both (marker IR bands depicted with C and G, respectively, in Figure 3c) are among these interfering compounds. The stratigraphic distribution of the compounds identified non-invasively, was examined through the study of micro-samples selectively taken from the mural and mounted in cross-sections. An example of the data set collected for a sample coming from the _Trinity_ background is provided in Figure 4. Firstly, the microscope examination (Figure 4b) allowed the distinction of two preparatory layers characterized by different properties and morphological aspect as clearly visible in the SEM-BSE images (Figure 4c). The micro-FT-IR measurements across the entire stratigraphy of the sample (Figure 4d) permitted the detection of gypsum exclusively in the plaster (layer 1) with a possible weak contribution of an organic material (carbonyl band between 1711 and 1750 cm\({}^{-1}\) localized in a thin line of layer 1--Figure 4e). On the top of the gypsum-based plaster, the identification of amide I band (1695-1615 cm\({}^{-1}\)) suggested the presence of a primer (layer 2) composed of a proteinaceous material and followed by a lime-based preparatory layer (layer 3). In the latter, the weak protein component observed (Figure 4e) is probably penetrated from the upper protein bound paint layer (layer 4) containing ivory black (see phosphate group bands between 1061 and 992 cm\({}^{-1}\)[22] in Figure 4e). A representative IR spectrum of layer 4 is shown in Figure S3. Concerning the lime-based preparatory layers, it is important to point out that in the cross-sections obtained from two samples coming from the upper part of the vault, an additional yellow line-based layer (Figure 5b, layer 4) is visible above the white lime-based layer (Figure 5b, layer 3). The yellow preparatory layer consists of a blend of yellow ochre and cadmium yellow mixed with calcium carbonate. The presence of cadmium yellow in this underlayer explains the element cadmium found in all the XRF spectra, a total of 39 spectra, acquired on the two angels in the upper part of the vault (Figure 2b), particularly where the color of the paint layer is a dark blue or black, i.e., not associable with a cadmium-based pigment. This yellow preparatory layer does not seem to be present in the rest of the _Trinity_. In the sample shown in Figure 5, over this yellow preparatory layer, the paint layer is visible in multiple applications (layer 5). The paint layers contain zinc white, yellow ochre, cadmium yellow, ivory black and an organic red pigment, all mixed with a protein-based binder. On the sample surface, the last layer (layer 6) is a non-original application of an acrylic resin. The cross-section shows that the yellow preparatory layer is broken and lifting, probably due to the application and the shrinkage upon drying of the acrylic surface coating. Overall, high protein amounts were found in correspondence with the paint layer in each _Trinity_ sample examined, confirming the data emerged from the in situ infrared investigations. Moreover, in addition to protein signals, most of the non-invasive FT-IR spectra contained the signal at ca. 1740 cm\({}^{-1}\) related to carbonyl stretching mode and sharp CH bands between 3000-2700 cm\({}^{-1}\) that suggest the possible presence of a lipid component.