## 3 Physico-Chemical Analyses
The investigations which were conducted on one of them, specifically the Kiev Folia, were performed directly in the Vernadsky National Library of Ukraine, and this included, first, the identification of the type of ink that was used in the text, which was iron gall ink [8]. This result was obtained by merging together the data of the XRF technique (showing a high number of counts in the quantity measure of elemental iron (Fe)) and Raman spectroscopy (Figure 3a,b). An FTIR analysis which was performed on the writings revealed the presence of calcium oxalates (Figure 3c), which were probably formed after the oxidative degradation of the ink components, which can occur due to a biological attack [15; 17]. As described in more detailed in Section 4.2.2, as a result of this particular study, the microorganisms that were detected with the most plausible capacities to produce calcium oxalate precipitation were the species of the genera _Staphylococcus_ and _Streptococcus_[8]. A high amount of calcium (Ca) was detected by the XRF method on the parchmentfolios. The complementarity of the approach revealed not only the presence of calcium carbonate (CaCO\({}_{3}\)) deriving from the manufacturing of the material, but also from the presence of calcium stearate (e.g., calcium soaps) which was probably formed during the depilation process by the reaction of the calcium in the lime bath and the lipidic content (subcutaneous fat) of the parchment [8]. This material was also found in another of the previously investigated Slavonic codices, the _Codex Assemanianus_. In fact, in this precious manuscript, calcium soaps and oxalates were also detected. Once again, further microbiological investigations were performed on this manuscript in order to better understand the conservation status of the item and support the hypothesis on the future risk of deterioration, as shown in the next sections [3]. Furthermore, all of the complementary methods that have been described and applied for the analysis of the mentioned manuscripts represent, in general, the optimal non-invasive tools for the identification of (I) inorganic and organic pigments that were used for illuminations in manuscripts (e.g., lapis lazuli, azurite, and indigo, lead white, vermilion, etc. ), and extenders (e.g., chalk or kaolinite), (II) materials that  
Figure 3: Analysis of black ink in the Kiev Folia performed with (**a**) XRF, showing its elemental composition with high counts in the quantity measure of iron (Fe) in comparison with the parchment (in blue), suggesting the presence of an iron-based ink. (**b**) Raman spectrum confirming the presence of an iron gall ink (green reference spectrum), and (**c**) rFTIR revealing the presence of calcium oxalate (green reference spectrum). were used for the preparation of the proteinaceous support (e.g., silicates due to the usage of pumice stone), as well as (III) substances that were applied during the later conservation processes (e.g., starch) [8; 28; 33]. Finally, it is important to emphasize that the combination of analytical techniques can render additional information about ancient manuscripts, as shown in the study of [PERSON] et al. [34]. The authors show how results that were obtained by scanning electron microscope imaging which was coupled with dispersive X-ray microanalysis, Raman microscopy and Fourier Transform Infrared Spectroscopy can reveal the presence of microobjects, which are both inorganic and organic, and are associated with the parchment fibers or in the inks and dirt that have accumulated over the centuries between the pages. These observations can reveal important information about the manufacture of ancient documents, about their history or about the causes of ageing and deterioration of the materials which they contain. The authors also argue that some chemical components, which are normally attributed to the manufacture of materials or inks, may instead have a particular origin and nature, and therefore, lead, if they are not properly highlighted, to some misdiagnoses.