Gas Chromatographic Analysis of Amino Acids as Ethyl Chloroformate Derivatives. Part 1, Composition of Proteins Associated with Art Objects and Monuments

Characterisation of proteinaceous binders in artistic paintings by chromatographic techniques

“…Unfortunately, markers are only available in a few cases and care must be taken in the interpretation, especially when protein mixtures are present. It is acknowledged that polysaccharidic gums, widely used as binders and adhesives in art, contain low contents of proteinaceous matter (about 3% in arabic gum) including hydroxyproline [23]. Thus, an identification based only on the presence of hydroxyproline could erroneously lead to inferring the presence of animal glue and to ignoring the presence of other proteins.…”

“…Quite often these data are not reported in the literature since no quantitative analysis of amino acids is performed. In any case, the available data [23,25,34,39,55] show average recoveries of 85 -96% for collagen, 64 -84% for egg, and 86 -89% for casein, thus highlighting that all the analytical techniques work well for pure, unpigmented samples of non-aged protein [23]. One specific problem encountered in the application of chromatographic diagnostic techniques to works of art is the lack of certified reference materials which guarantee the quality of the data.…”

“…To reduce the risks of contamination connected with the addition of HCl solution into the sample, hydrolysis in the vapour phase has often been adopted [23,25,37,39]. The performance of vapour acidic hydrolysis has been significantly improved by carrying out the hydrolysis with 6 M HCl in a microwave oven under nitrogen atmosphere, at 8 atm and 1608C for 40 min [25].…”

“…Their mass spectra show a very simple fragmentation pattern and are normally dominated by a large abundance of ions with m/z equal to M -57 + (loss of a tert-butyl group), which enable molecular weight determination and are very suitable for performing quantitative analysis by Selected Ion Monitoring. c) Derivatisation with ethyl chloroformiate in ethanol/ water/pyridine [23,24,29,61]. The reaction, which takes place in one step, in only a few seconds and in the presence of water, acts on both amino and hydroxyl groups.…”

“…Acidic hydrolysis, the most common method, has been performed under several experimental conditions with reaction times between 1 and 24 hours. Acidic hydrolysis performed with 6 M HCl at 1108C, under vacuum or in an inert atmo-sphere of nitrogen or argon, is essentially the most common method for hydrolysing proteins and peptides [3,23,24,27] in paint samples. Depending on the conditions, the recovery is the result of a compromise between an efficient hydrolysis of the most stable peptidic linkages (ileile, val-val, ala-ala, ile-ala) and the minimum losses of the less stable amino acids.…”