Characterization of Proteinaceous Binders in Wall Painting Samples by Microwave-Assisted Acid Hydrolysis and GC-MS Determination of Amino Acids

The analysis of naturally and artificially aged protein‐based paint media using Raman spectroscopy combined with Principal Component Analysis

“…A particular strength of chromatographic techniques is that, with ageing, a few changes are associated with the concentration of detected amino acids, which form the basis of the analysis of proteins. 26,27 In contrast, with Raman spectroscopy, light-induced changes in artificially aged egg white and egg yolk have been observed with a reduction in intensity of aromatic amino acid vibrations in the fingerprint region, which has also been confirmed with excitation emission fluorescence spectroscopy 28 and analogous studies using MALDI-MS. 29 However, binding media from animal tissues and milk have not been examined and furthermore, the analysis of C-H stretching between 2700 and 3100 cm 1 , for the differentiation among mixtures of binding media (both naturally and artificially aged) as well as the assessment of ageing of specific binding media has not been undertaken.…”

GC/MS in the Characterisation of Protein Paint Binders

“…• Silylation with formation of trimethylsilyl (TMS) derivatives [55,75] or tert-butyldimethylsilyl (TBDMS) derivatives [47,48,[85][86][87] has the advantage of the simultaneous derivatisation of acidic and aminic functions. However, the absence of humidity is critical also in this method; indeed, water would degrade the silylating reagent.…”

“…The range of chromatographic techniques applied to the characterisation of the proteinaceous constituents in paint includes paper chromatography [32,33], thin-layer chromatography (TLC) [34][35][36], ionic exchange chromatography [37,38], reversed phase high-performance liquid chromatography with pre-column derivatisation [14,[39][40][41][42][43][44][45], gas chromatography [7,15,16,30,[46][47][48][49][50][51][52][53][54][55][56][57], and pyrolysis combined with gas chromatography-mass spectrometry (Py-GC/MS) [58][59][60][61][62][63][64][65][66]. Gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS) are among the most commonly used techniques for the identification of proteinaceous paint media, and they generally give more accurate and reliable results than other methods due to their high specificity and sensitivity [5,11,67].…”

“…Two typical methods used to achieve the hydrolysis of peptidic bonds are enzymatic and chemical catalysis [73]. The reaction times for enzymatic hydrolysis are long and typically lie in the range of 4-8 h [47]. Additionally, they demand purification procedures to get rid of the excess enzyme that could interfere in the protein identification.…”

“…Hydrolysis in the vapour phase has often been adopted as a tool for decreasing the chances of contamination related to the addition of HCl solution into the sample [14,15,42,47]. Microwave assisted hydrolysis using 6 M HCl under nitrogen atmosphere, at 8 atm and 160°C for 40 min (250 W) [47] or using 10 M HCl at 240 W for 90 min [77] has greatly improved the results of vapour acidic hydrolysis leading to either comparable or enhanced yields to those recovered with the classical HCl hydrolysis conditions (6 N HCl, 110°C, 24 h) while dramatically reducing the digestion time. The additional electromagnetic energy improves the efficiency of the hydrolysis of peptide bonds and reduces the amino acid losses due to oxidation and degradation which are characteristic of methods which require longer reaction times.…”