# Organic Materials Used for Giant Buddhas and Wall Paintings in Bamiyan, Afghanistan
## 4 Discussion  
Our various complementary micro-analyses revealed the presence of a large variety of organic components in different layers of samples taken from the surfaces of the Eastern and Western Giant Buddhas and the Bamiyan wall paintings. In the deepest layers (transparent sizing layer), SR-uFTIR highlighted the presence of proteins. GC-MS analyses on six samples from the EGB and seven samples from the WGB confirmed that the paintings contained proteinaceous binding media. Notably, samples from the EGB (BMM191) and WGB (BMM199, 201) contained cow glue, including cow skin glue (cattle hide) (BMM201) identified through the presence of type III collagen by nano-LC/ESI-MS/MS. Unexpectedly, horse glue was detected in the wall paintings of Cave N(a) (BMM194) by LC-MS in MRM mode, as described in Section 3.5, and could have been used as a sizing agent. However, this is a rare example from Bamiyan. The recently developed analytical protocol with LC-MS (Shi2019; Shi2019) is a rather convenient and quick method for identifying specific species of animals, primarily in the leather industry, and is a powerful tool for paint analysis. However, casein (cow milk) was identified in the EGB samples and BMM201 (WGB) and could have been used as a sizing agent under the painting layers. Casein was also identified in samples from the wall paintings of Cave A (BMM061) and Cave M (BMM078) by LC-MS/MS. The presence of casein could support a historical recipe recorded in the _citrasatra_ (a technical and religious treatise on Buddhist paintings in India), which mentions the use of cow milk on earthen walls (Shi2019). On the contrary, the ELISA method revealed casein in all samples, plant gum in three samples, and tragacanth in one sample. These could stand as evidence that, in each painting, plant gums and tragacanth served as binding media and the protein (casein) as a sizing agent. Plant gums and tragacanth seem to be readily detected owing to the method's high sensitivity with regard to minor organic materials that may not be identified using LC-MS or GC-MS, although this method has a rather narrow range of candidates for testing due to its methodological limitations. Below the proteinaceous sizing layer, SR-uFTIR sometimes detected the presence of a natural resin. However, GC/MS did not confirm resin in these samples but detected trace amounts of pine resin in other caves. Above the sizing layers, SR-uFTIR analyses  
Figure 10: ESI-MS/MS spectrum of the precursor ion peak at \(m/z\) 634.6 (\(z=2\)). In the sequence of YGLYLEQ\({}^{\ast}\)LLR, Q\({}^{\ast}\) represents Glu (E) modified from Gln (Q) by the deamidation associated with aging. revealed that the white ground layers (similar to _imprimatura_) contain partially saponified fatty esters (or the presence of lead soaps) mixed with lead white pigment (cerussite and hydrocerussite). A similar saponified fatty medium was also detected in some painted layers. Thanks to GC-MS, drying oils (vegetable oils) were more precisely identified in the wall paintings of 12 caves. Some other fatty acids were also detected, which could be related to egg or other sources. Considering the recurrent presence of lead soaps, their homogeneous distribution in the ground and some painted layers, and the good conservation state of these layers, the assignment of lead soaps to an intentional synthesis should not be neglected [50]. In Shosoin Repository, Nara, Japan (8 th century AD), a technique named _mitsuda-e_, based on the name _mitsudaso_, which refers to litharge (PbO), describes the mixing of PbO with vegetable oil. This recipe is assumed to originate from Persia [51]. It may be a link between historical painting techniques and the Bamiyan paintings. Oil-based mordants were also found under metal foils used as surface decorations (BMM178, QJM06). Finally, numerous surface areas contained oxalates (mainly calcium and sometimes copper), which could be degradation products of organic components that form lightly colored films on the paint surface.