SUPPLEMENTARY MATERIALS
1 PY-GC/MS
The pyrolysis of the samples analyzed confirms the presence of the organic materials already
identified by GC/MS and summarized in Table S 1.
Table S 1 Summary of results from the Py-GC/MS analysis
Samples
Animal glue
Pyrrole
PFX 8 PR
PFX 15
SX 5 B
SX 5 M
SX 8 PL
SX 10 B
SX 10 M
SD 2
X
X
X
X
X
X
X
X
Egg
Markers
Hexadecanonitrile
Octedecnonitrile
X
X
X
X
-
Saccharide materials
Levoglucosan
Xylofuranose
X
X
X
X
X
2 GC/MS
The quantitative determination of amino acids, aldoses and uronic acids, aliphatic mono- and
dicarboxylic acids is performed by using standard solutions, building calibration curves, and
evaluating daily recoveries. Running blanks of the procedure highlighted a low level of
contamination. The detection limit (LOD) and the quantitation limit (LOQ) of amino acids,
aldoses, uronic acids, and fatty and dicarboxylic acids were calculated. At a statistical
significance level of 0.05, the LODs and LOQs obtained of the proteinaceous, glycerolipids
and saccharide materials were as follows:
Proteinaceous materials: LOD: 0.2 μg; LOQ: 0.3 μg
Glycerolipids: LOD: 0.3 μg; LOQ: 0.7 μg
Saccharide materials: LOD: 0.3 μg; LOQ: 0.5 μg.
2.1 Proteinaceous fraction
The proteinaceous content of all the samples analyzed resulted to be higher than LOQ (0.3
µg) and Table S 2 summarizes their amino acidic percentages.
Table S 2 Relative percentage of amino acid content and total amount of proteins in the
analyzed samples. Ala: alanine, Gly: glycine, Val: valine, Leu: leucine, Ile: isoleucine, Ser:
serine, Pro: proline, Phe: phenylalanine, Asp: aspartic acid, Glu: glutamic acid, Hyp:
hydroxyproline
Amminoacidic
Sample
Ala
Gly
Val
Leu
Ile
Ser
Pro
Phe
Asp
Glu
Hyp
YX 1 I
9.9
14.9
9.1
13.6
6.4
4.0
5.9
5.5
13.5
17.4
0.0
0.3
YX 1 PR
9.9
18.3
4.6
6.7
3.2
4.6
11.5
4.4
9.9
17.0
9.7
9138.1
PFX 2
8.8
10.8
8.9
11.4
6.3
4.7
6.8
5.3
20.1
17.0
0.0
0.3
PFX 8 T
10.4
29.8
3.3
6.7
2.9
2.5
8.2
3.5
11.6
14.4
6.6
1.2
PFX 11 T
17.5
44.2
4.1
4.9
2.7
1.5
13.8
1.5
4.7
4.8
0.3
1.8
PFX 11 PL
13.3
33.4
3.5
4.8
2.4
2.3
13.2
2.6
8.9
14.0
1.7
1.4
SX 4
7.9
17.7
3.9
8.5
4.0
8.0
10.0
5.2
19.7
14.2
0.9
0.6
SX 5 T
8.9
25,7
2.7
4.4
1.9
4.8
12.1
3.3
10.0
21.0
5.1
14.4
SX 6
4.9
9.70
1.8
3.6
1.5
9.4
3.0
3.7
16.7
22.7
23.0
10.4
SX 8 T
1.0
39.1
5.9
9.9
4.5
4.0
16.6
2.9
6.5
5.6
4.0
2.1
SX 9 B
3.0
31.3
2.9
4.2
2.1
5.7
8.5
3.1
12.2
18.9
8.2
4.4
SX 9 S
14.7
30.5
8.7
9.8
5.1
5.2
14.5
1.4
31.0
2.8
4.1
0.7
SX 10 M
9.0
23.5
5.5
9.4
4.5
3.3
12.8
5.7
14.3
11.4
0.8
0.5
SX 10 B
8.4
17.3
30.8
9.0
4.1
2.6
7.1
4.1
7.7
7.1
1.9
1.0
SD 3
2.0
25.2
6.4
9.3
5.3
2.6
18.4
4.3
9.2
14.4
2.6
0.7
SD 4
7.2
15.6
20.2
4.5
2.2
4.6
7.8
2.6
7.1
15.2
4.0
16.5
content (µg)
Figure S1 shoes the loading plot of the reference samples used as database for the PCA
analysis of the amminoacidic profiles obtained from the samples. The loading plot
highlights the important role that each amino acid plays in the separation of clusters.
Figure S1 Loading plot of the reference samples
2.2 Saccharide fraction
Analyses results show that 3 samples present a content of saccharide material higher than
LOQ (0.5 µg) while sugars content of 4 samples is between the LOD (0.3 µg) and LOQ.
Table S 3 summarizes the glycosidic profiles of the seven samples whose saccharide material
is higher than the LOQ and the presence/absence of the singular sugars in those samples
showing a saccharide content between the LOD and LOQ.
Table S 3 Relative percentage of monosaccharides and uronic acid content, total amount of
saccharides in the analyzed samples. Xyl: xylose, Ara: arabinose, Ram: rhamnose, Fuc:
fucose, Gal ac: galacturonic acid, Glu ac: glucuronic acid, Glu: glucose, Man: mannose, Gal:
galactose
Saccahride
Sample
Xyl
Ara
Ramn
Fuc
Galact ac
Glu ac
Glu
Man
Galact
YX 1 I
18.5
26.4
0.6
0.0
0.0
0.0
26.8
7.8
19.9
44.3
PFX 2
20.7
14.9
1.4
0.7
0.0
0.0
45.8
9.3
7.2
0.9
SX 4
14.3
10.8
2.1
1.0
0.0
2.7
33.4
17.2
18.5
0.5
SX 5 T
X
X
X
X
-
X
X
X
X
0.4
SX 9 B
X
X
-
-
-
-
X
X
X
0.4
SD 3
X
X
X
X
-
-
X
X
X
0.3
SD 4
X
X
X
X
-
-
X
X
X
0.3
X= presence of sugar
content (µg)
2.3 Lipid fraction
Lipid chromatographic profiles reveal the absence of the markers of terpenic resins, shellac
and natural waxes. In quite all samples analyzed, the amount of lipid material is generally
low although above LOQ (0.7 µg).
Table S 4 Relative percentage of lipid, total amount of lipid and their characteristic ratios in
the analyzed sample. Lau = lauric acid, Sub = suberic acid, Aze = azelaic acid, Mir = myristic
acid, Seb = sebacic acid, Palm = palmitic acid, Oleic = oleic acid, Stear = stearic acid. A/P:
azelaic acid over palmitic acid ratio, P/S: palmitic acid over stearic acid ratio, O/S oleic acid
over stearic acid ratio, ΣD: sum of dicarboxylic acids
Sample
Lau
Sub
Aze
Mir
Seb
Palm
Oleic
Stear
YX 1 I
3.0
0.1
1.4
2.8
0.4
30.2
27.3
34.0
YX 1 PR
2.9
0.5
0.8
2.2
0.2
26.9
32.2
Lipid
A/P
P/S
O/S
%ΣD
3.7
0.0
0.9
0.8
2.6
34.3
1.9
0.0
0.8
0.9
1.6
content (µg)
0.7
1.1
2.7
0.3
29.3
34.5
28.3
0.0
1.0
1.2
2.0
PFX 8 T
3.1
1.1
5.7
5.1
0.5
0.9
2.9
0.5
30.6
11.8
0.0
0.6
0.3
1.9
PFX 11 T
12.4
1.4
15.2
0.8
0.0
23.3
4.0
43.0
47.7
3.5
0.5
0.5
0.1
16.5
PFX 11 PL
20.1
0.4
1.6
1.4
0.3
23.7
17.6
34.9
1.2
0.1
0.7
0.5
2.3
SX 4
12.4
3.4
3.1
4.5
1.4
29.8
18.2
37.2
1.2
0.1
0.8
0.2
7.9
PF X 2
SX 5 T
1.1
1.1
2.9
3.8
6.0
37.6
0.8
46.6
4.4
0.1
0.8
0.0
10.1
SX 8 T
16.0
0.0
2.0
3.8
0.3
32.1
10.9
34.9
1.3
0.1
0.9
0.3
2.3
SX 10 M
1.8
4.5
7.3
3.3
1.2
38.4
1.5
42.0
0.6
0.2
0.9
0.0
13.0
SX 10 B
1.0
10.0
36.7
1.8
9.9
18.9
1.8
19.9
1.4
1.9
1.0
0.1
56.6
62.5
1.0
0.0
0.5
0.1
1.2
SD 4
2.5
0.5
0.5
1.2
0.1
29.5
3.1
3 Gilding inorganic materials: combined use of SR micro FT-IR and SR micro XRF
Micro XRF results highlighted the compositional difference between the adhesive layer
(layer II), characterized by the presence of Mn, Pb, Ca and P, and the white ground layer
(layer IV), containing Si, Al, K, Ca and Ti. The detection of Si, Al and K in the white ground
layer is in agreement with the presence of a clay identified as kaolinite thanks to the
octahedral coordination external O-H absorption peaks (3697, 3669 and 3654 cm-1) in the
FTIR spectrum (Fig. 6 section 3.3.).
The distribution of Fe, in an area between the adhesive layer and the preparation layer, is in
agreement with the red layer that could be observed in the cross-section (Fig. 2 i) and
particularly highlighted under UV light (Fig. S 2 c), The presence of Fe and Mn in the red
and adhesive layers may point to the use of iron based pigments (Fe2O3 or FeOOH) and
MnO2, in agreement with the use of a natural earth named umber [34] as red-orange pigments
added to the organic adhesive of the gold foil. In previous work on the mural paintings in the
Temples under Chieftain Lu in Liancheng (Gansu Province, China) an adhesive layer made
of a binder admixed with red iron oxide was also found under the gold leaf [33]. In the
literature, iron based pigments, red lead or other red pigments were commonly used in
gilding samples aiming at enhancing the color of gold [19].
Pb was identified into the adhesive layer. The presence of the OH absorption band around
3538 cm-1 (Fig. 6 section 3.3.) combined with strong absorption of COO- asymmetric
stretching vibration around 1400 cm-1 supports the identification of hydrocerussite
(Pb3(CO3)2(OH)2). It has been previously established that lead white was used either as the
major constituent or mixed with other colored pigments to produce different hues in the
mural paintings of the Five Northern Provinces’ Assembly Hall [19].
The presence of the characteristic OH stretching bands at 3544 and 3404 cm-1, as well as the
OH bending band at 1620 cm-1, is in agreement with the presence of gypsum (CaSO4·2H2O)
locally present in the adhesive layer.
The interpretation of the presence of phosphorous (P) in layer II and III from the XRF maps
is not straightforward. P has been traditionally linked to the presence of bone black
(hydroxylapatite), resulting from burning animal bones [19]. However, we cannot confirm its
presence by any other means. The use of animal glue as binder, extracted from boiling animal
bones, could be a possible explanation to the P distribution though this hypothesis would
need further investigation to be confirmed.
Fig. S2 (a) SR μXRF maps at 7.2 keV Mn, Ag, Ca, Au, Cl, Al, Fe, K, Pb, P, Si and S
acquired for the entire sample with 1×1 μm2 pixel size; (b) scheme of the sample build-up;
(c) UV image of the cross-section of sample SD 2