# Testing Clean-Up Methods for the Quantification of Monosaccharides and Uronic Acids
## 3 Results
The ATR-FT-IR analysis allowed us to confirm the expected high level of inorganic material in the sample and to determine it. In particular, the presence of a clay mineral, kaolinite (Al2 Si2O5(OH)4), was identified by the characteristic bands in the mid-IR range of 3686-3619 cm\({}^{-1}\) as well as at 1029, 1007, 915, 784, and 750 cm\({}^{-1}\) (Figure 5) [32,33]. The presence of gypsum (CaSO4-2H2O) was also assessed on the basis of typical bands at 595, 664, 1618, 3398, and 3529 cm\({}^{-1}\). Figure 3: Recoveries, expressed as a percentage with respect to the concentration of starting solution, reported for each fraction of eluate analyzed, relative to the clean-up test results (Experiment B) using a Macherey-Nagel(tm) Chromabond(tm) NH2 Polypropylene SPE column applied to a standard solution of 7 monosaccharides and 2 uronic acids (each at 10 ppm). The experiment was conducted in triplicate and the error bars represent the confidence interval. Given the high content of inorganic fraction, the sample is a suitable candidate to test the effectiveness of the optimized purification method. The GC-MS chromatogram in SIM mode of the mural paint sample after the purification step on a Macherey-Nagel(tm) Chromabond(tm) NH2 Polypropylene SPE column is shown in Figure 6, and the concentration of monosaccharide and uronic acids present is reported in Table 4.  
Figure 4: Recoveries (expressed as a percentage with respect to the concentration of starting solution) are reported for each fraction of eluate collected from the analysis of a standard solution of 7 monosaccharides and 2 uronic acids (each at 10 ppm). The clean-up test selected, makes use of a Macherey-Nagel(tm) Chromabond(tm) NH2 Polypropylene SPE column (Experiment C). The experiment was conducted in triplicate and the error bars represent the confidence intervals. Figure 5: ATR-FT-IR spectrum of the preparation layer of a mural painting sample, recorded with 16 scans at 4 cm\({}^{-1}\) spectral resolution. The bands associated with the presence of kaolinite (750, 784, 915, 1007, 1029, 3616, and 3686 cm\({}^{-1}\)) are highlighted with the asterisk (*), while those due to gypsum (595, 664, 1618, 3398, and 3529 cm\({}^{-1}\)) are labeled with the symbol (§). The optical microscope image of the sample is depicted on the top. Since the presence of inorganic ions seems to have a considerable influence on the derivatization reaction [30], the purification effectiveness was evaluated for the archaeological sample. The recovery of mannitol, the internal standard of derivatization (I.S. ), was calculated as the ratio between the initial I.S. concentration and that obtained from the integration of the chromatographic peak of the derivatized I.S. The I.S. recovery for the paint sample rich in the inorganic sample was 0.82, demonstrating the achievement of good efficiency of derivatization, and the effective removal of the interfere. On the basis of the literature, the evaluation of the relative content of saccharides and uronic acids can provide information on the vegetal gum employed [11]. The most abundant saccharides present in the hydrolysate sample are arabinose and the two uronic acids. Given the absence of fucose and rhamn, the profile appears to be comparable to that of a fruit tree vegetable gum.