GC-MS identification of proteins in wall painting samples: A fast clean-up procedure to remove copper-based pigment interferences

A new approach was explored to purify proteins in a multi-step procedure for the characterisation of proteinaceous materials (casein, animal glue, and egg) in artwork samples by gas chromatography-mass spectrometry. High concentrations of inorganic salts, such as azurite, have been found to impair the determination of protein via amino acid analysis. The effect of varying concentrations of copper-based pigments on the quantification of amino acids was evaluated through the analysis of replica paintings prepared with the three types of proteinaceous materials. Glycine, aspartic and glutamic acids are the amino acids most affected by the presence of copper salts. In the case of high concentration of salts, this interference hampers the correct identification of the proteins. To eliminate the inorganic salts, a C18 pipette tip was used to clean-up the ammonia extracts before the acidic hydrolysis step. The clean-up procedure allows us to prevent the influence of the inorganic salts and thus allows correct protein identification, though the quantitative recovery of proteinaceous material is quite low. The effectiveness of the optimised procedure was evaluated by analysing samples from two Italian wall paintings from the 13th and the 14th centuries. Without the clean-up it would not have been possible to detect the presence of a mixture of egg and animal glue in one case, and that of egg in the other one.     

Animal glues in mixtures of natural binding media used in artistic and historic objects: identification by capillary zone electrophoresis

Animal glues were often used in historic and artistic objects, e.g. as paint ground, as binders for pigments, or as adhesives. The sources were egg, casein, or different collagens. For restoration and conservation purposes it is important to know which kind of animal glue a museum object contains. Capillary electrophoresis can deliver such information, because it enables differentiation among the three proteinaceous glue classes according to their different amino acid patterns after hydrolysis. This work deals with the most relevant problem in practice, whether this identification is obstructed by the presence of other binders, with which they are mixed in many real samples; in particular, interference from plant gums and drying oils was investigated. Capillary electrophoresis of the hydrolysates (after reaction with 6 mol L^–1 HCl) was performed with an acidic background electrolyte consisting of chloroacetic acid (51.9 mmol L^–1) adjusted with LiOH to pH 2.26. The underivatised analytes were detected with a contactless conductivity detector. It was found that the constituents of the plant gums (monosaccharides) or drying oils (long-chain fatty acids and short-chain dicarboxylic acids) never interfered with identification of the animal glues, as shown for artificial mixtures of the different binders even at tenfold excess over the animal glue, and for egg tempera samples. The method was used to identify the filling material from a statue from the eighteenth century.     

Synthesis and antimicrobial activity of some netropsin analogues

Nine novel lexitropsins were synthesized by linking two netropsin-like moieties through three different dicarboxylic acids; 9,10-dihydro-2,7-phenanthrenedicarboxylic acid; [(3-[[(carboxymethyl)amino]carbonyl]benzoyl)amino]acetic acid and indole-2,5-dicarboxylic acid. The netropsin residues were modified by the use of N-isopentylpyrrole, 5-methylthiophene or 5-isopropylthiazole heterocyclic building blocks in place of the usual N-methylpyrrole. The compounds were tested against five gram-positive bacteria: Staphylococcus aureus, Streptomyces faecalis, methicillin resistant Staphylococcus aureus, Enterobacter cloacae, Mycobacterium fortuitum, three gram-negative bacteria: Klebsiella aerogenes, Proteus vulgaris, Escherichia coli and three fungi: Aspergillus niger, Candida albicans and Aspergillus nidulans. Some of the compounds showed significant inhibitory effects on the growth of the microorganisms.     

氨基酸分析法研究蒙古国博格达汗宫建筑彩画的胶料种类

为了鉴定博格达汗宫建筑彩画使用的胶料,采用氨基酸分析法测定了古代常用胶料(鸡蛋、动物胶及奶类)的氨基酸组成比.分析3类胶料,发现各胶料的氨基酸组成特点,其中动物胶含羟脯氨酸,且甘氨酸平均含量高达20.45%;鸡蛋中天冬氨酸与脯氨酸的比值较高,平均值为2.29;奶类中谷氨酸与丙氨酸的比值较高,平均值为5.01.在此基础上,分析了蒙古国博格达汗宫建筑彩画样品,对样品的氨基酸组成比进行主成分分析,确定了样品胶料的种类.同时,以动物胶特有的氨基酸--羟脯氨酸(含量均值13.6%)为研究对象,估算出文物样品使用胶料的配方为鸡蛋胶原/动物胶胶原(1.5∶ 1),得出了蒙古国博格达汗宫建筑彩画是采用以鸡蛋为主、动物胶为辅的混合胶料的结论.     

Pitfalls in drying oils identification in art objects by gas chromatography

Drying oils identification in art objects is an important step in the scientific investigation of the artifact which provides conservators and art historians with valuable information concerning materials used and painting techniques applied. The present communication is devoted to pitfalls and troubleshooting in drying oils identification by means of GC-MS analysis of fatty acids composition in a microsample of an art object. We demonstrate that in the case of nonlinear instrument response the ratios of palmitic to stearic (P/S), distinctive for each oil type and used for drying oil identification, depend on sample dilution so that different dilutions of the same sample can give different P/S ratios. This phenomenon can hinder drying oil identification and lead to erroneous interpretations. This is an important observation as nowadays very often the P/S ratio is calculated from the corresponding peak area ratios or by the use of one-point calibration method. In these approaches, the linearity of the instrument response is not controlled and ensured. In the case analyzed, the nonlinear instrument response was attributed to incomplete sample evaporation in the injector. Packing of the glass liner with deactivated glass wool improved the sample evaporation and ensured the linearity of the instrument response and independence of the P/S ratio from sample dilution.     

Identification of garlic in old gildings by gas chromatography-mass spectrometry

The proteinaceous content of garlic (Allium sativum) was characterised according to its amino acid composition by using a gas chromatography-mass spectrometry (GC-MS) analytical procedure. The procedure was tested on fresh and aged garlic samples as well as on reference gilding specimens prepared according to old recipes. The proteinaceous pattern showed a characteristic distribution of amino acids with glutamic acid being the major component. The average amino acidic composition was: glutamic acid (Glu; 29%), aspartic acid (Asp; 17%), serine (Ser; 11%), alanine, glycine, valine, leucine, lysine and phenylalanine (Ala, Gly, Val, Leu, Lys and Phe; 5-6%), isoleucine, proline and tyrosine (Ile, Pro and Tyr; 2-3%), methionine and hydroxyproline (Met and Hyp; 0.5%). In order to distinguish this material from animal glue and egg, which are the other proteinaceous media commonly used in gilding techniques, a database of amino acid percentages of the three proteins was built up and submitted to principal component analysis. Three separate clusters were obtained, allowing the protein identification. The application of the procedure on several gilding samples from Italian wall and easel paintings (13th-17th century) permitted to evidence the use of garlic as a gluing agent.     

Biotransformation of citrus aromatics nootkatone and valencene by microorganisms

Biotransformations of the sesquiterpene ketone nootkatone from the crude drug Alpiniae Fructus and grapefruit oil, and the sesquiterpene hydrocarbon valencene from Valencia orange oil were carried out with microorganisms such as Aspergillus niger, Botryosphaeria dothidea, and Fusarium culmorum to afford structurally interesting metabolites. Their stereostructures were established by a combination of high-resolution NMR spectral and X-ray crystallographic analysis and chemical reaction. Metabolic pathways of compounds and by A. niger are proposed.     

Influence of harvesting time on composition of the essential oil of Thymus capitatus (L.) Hoffmanns. & Link. growing wild in northern Sicily and its activity on microorganisms affecting historical art crafts

One of the main factors affecting historical art crafts material is the biodeterioration performed by bacteria and fungi, in archives, museums or private collections. Several microorganisms cause degradation to the natural organic material such as fibers, woods, and dyes as well as to stone objects. These alterations produce deterioration of physical, chemical, mechanical and esthetic properties. Consequently, in this publication, we report the high antibacterial and antifungal activities of wild thyme essential oil, oil that can be used as an alternative natural tool in the fight against microorganisms affecting historical art crafts.Essential oil of the Thymus capitatus growing wild in northern Sicily has been extracted by hydrodistillation from aerial parts collected at different growth times. The constituents of the essential oil have been characterized by gas chromatography (GC) and GC–mass spectrometry.Fifty-five compounds have been identified. Essential oils were characterized by a high content of carvacrol (81.2–14.2%), γ-terpinene (34.4–2.6%) and p-cymene (22.8–5.0%) of the total oil content. Essential oil yield and composition vary throughout the vegetation time of the plant. The best time to harvest this species of thyme, for phenol content, is during or immediately before the full bloom. The related oils (Tc2 and Tc3) showed a good antibacterial activity against Bacillus subtilis and Staphylococcus epidermidis and excellent antifungal properties against Fusarium oxysporum and Aspergillus niger.     

Evaluation of preservative effectiveness of liquid crystalline systems with retynil palmitate by the challenge test and D-value

The objective of this work was to evaluate the preservative effectiveness of liquid crystalline systems containing retynil palmitate (RP) by the challenge test (CT) and D-value. A system was developed containing water, silicon glycol copolymer, and polyether functional siloxane with 1% RP added. The analyses were carried out by methods in the U.S. Pharmacopeia (USP 31, 2008) using the microorganisms Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Aspergillus niger. The CT showed that after 7 days, all microorganisms were eliminated except A. niger, which maintained viability for at least 28 days after inoculation. Moreover, the microorganisms E. coli, P. aeruginosa, S. aureus, C. albicans, and A. niger presented different growth behaviors, evidenced by differences among the D-values calculated. It was concluded that the CT and D-value were efficient methods for evaluation of the preservative property of these formulations.     

Plasma-enhanced copolymerization of amino acid and synthetic monomers

In this paper we report the use of plasma-enhanced chemical vapor deposition (PECVD) for the simultaneous deposition and copolymerization of an amino acid with other organic and inorganic monomers. We investigate the fundamental effects of plasma-enhanced copolymerization on different material chemistries in stable ultrathin coatings of mixed composition with an amino acid component. This study serves to determine the feasibility of a direct, facile method for integrating biocompatible/active materials into robust polymerized coatings with the ability to plasma copolymerize a biological molecule (L-tyrosine) with different synthetic materials in a dry, one-step process to form ultrathin coatings of mixed composition. This process may lead to a method of interfacing biologic systems with synthetic materials as a way to enhance the biomaterial-tissue interface and preserve biological activity within composite films.     

GC-MS characterization of proteinaceous and lipid binders in UV aged polychrome artifacts

This paper presents the most significant results obtained from the characterization of protein and lipid binders in a broad range of reference paint materials prepared and stored at Opificio delle Pietre Dure in Florence (Italian Ministry of the Cultural Heritage, Italy). The amino acid distribution for protein binders and the fatty acid distribution for lipid binders were determined by gas chromatography-mass spectrometry before and after being artificially aged through exposure to UV light at 254 nm or 366 nm for different time periods in a climatic chamber (20°C and 80% RH). The results were compared with those relevant to old paintings of different ages, and showed that UV light aging does not significantly affect the amino acid profile of protein binders. Consequently, protein binders in old paintings can be reliably identified by comparing the amino acid composition with that of reference paint materials which have not been aged. However, the composition of lipid binders is substantially affected by UV irradiation, leading to a lowering of oleic acid and the formation of azelaic acid and other dicarboxylic acids including oxalic acid. An oleic to stearic acid ratio of less than 0.5 was observed in all the samples of works of art, and this parameter can be used to evaluate the extent of the artificial aging process. The formation of oxalic acid was also observed starting with pure unsaturated fatty acids, thus supporting the chemical origin of oxalate patina.     

Biotransformation of aristolane- and 2,3-secoaromadendrane-type sesquiterpenoids having a 1,1-dimethylcyclopropane ring by Chlorella fusca var. vacuolata, mucor species, and Aspergillus niger

Biotransformation of the aristolane-type sesquiterpene hydrocarbon (+)-1(10)-aristolene (1) from the crude drug Nardostachys chinensis and of the 2,3-secoaromadendrane-type sesquiterpene lactone plagiochilide (2) from the liverwort Plagiochila fruticosa by three microorganisms, Chlorella fusca var. vacuolata, Mucor species, and Aspergillus niger was investigated. C. fusca var. vacuolata and Mucor sp. introduced oxygen function into the cyclohexane ring of aristolene while A. niger oxidized stereoselectively one methyl of the 1,1-dimethyl group on the cyclopropane ring of aristolanes and 2,3-secoaromadendrane to give C-12 primary alcohol and C-12 carboxylic acid. The possible metabolic pathway of the formation of new metabolites is discussed. The stereostructures of new metabolites were established by a combination of NMR spectroscopy including HMBC and NOESY, X-ray crystallographic analysis, and chemical reaction.     

Application of the Kohonen artificial neural network in the identification of proteinaceous binders in samples of panel painting using gas chromatography-mass spectrometry

Historically, three types of proteinaceous matter--casein, egg and animal glue--were used as binders for pigments or as adhesives in easel and wall painting. The relative percentage content of alanine, glycine, valine, leucine, isoleucine, serine, tyrosine, phenylalanine, aspartic acid, glutamic acid, lysine, methionine, proline and hydroxyproline, as determined by GC-MS, is used for binder identification. In this paper we analyse the viability of a multivariate modelling using Kohonen's neural network to characterise the wood adhesive in 16 old samples from Italian panel paintings of the 12-16th centuries. As a training set we use the amino acid composition of 141 samples contributed by the Opificio delle Pietre Dure of Florence (Cultural Heritage Ministry, Italy). Of the 141 samples, 113 were used to train the Kohonen neural network and the remaining 28 as the evaluation set. A specificity and sensitivity of 100% was achieved in training and 92-100% in prediction depending on the assignation criteria employed. The neural network thus trained and evaluated was applied to the old samples, achieving identification of all of them. In addition, the map obtained for each amino acid provides relevant information as to its importance in the characterisation of the sample.     

Application of gas chromatography-mass spectrometry analysis to the study of works of art: paint media identification in polychrome multi-material sculptures

A new gas chromatographic-mass spectrometric procedure for characterizing both drying oils and proteinaceous binders in samples of painted artworks has been developed. Furthermore, a new analytical procedure for analysis of polysaccharide materials through identification of the monosaccharide constituents is proposed. The methods have been applied to characterizing binding media of the different layers of the polychrome surface in the multi-material sculptures from the sanctuary of Santa Maria delle Grazie in Mantova, Italy. It was found that animal glue was the main binder in the priming layer of all statues, whereas more complex mixtures were used in the paint layers. Generally, a drying oil was present, most often linseed oil alone or in combination with other organic binders.     

Tracing the biological origin of animal glues used in paintings through mitochondrial DNA analysis

We report the development of a suitable protocol for the identification of the biological origin of binding media on tiny samples from ancient paintings, by exploitation of the high specificity and high sensitivity offered by the state-of-the art DNA analysis. In particular, our aim was to molecularly characterize mitochondrial regions of the animal species traditionally employed for obtaining glues. The model has been developed using aged painting models and then tested to analyze the organic components in samples from the polychrome terracotta Madonna of Citerna by Donatello (1415-1420), where, by GC-MS and FTIR spectroscopy, animal glues and siccative oils were identified. The results obtained are good in terms of both sensibility and specificity of the method. First of all, it was possible to confirm that Donatello used animal glue for the preparation of the painted layers of the Madonna of Citerna and, specifically, glue derived from Bos taurus. Data obtained from sequencing confirm that each sample contains animal glue, revealing that it was mostly prepared from two common European taurine lineages called T2 and T3. There is one remarkable exception represented by one sample which falls into a surviving lineage of the now extinct European aurochs.     

BIODEGRADATION OF REGENERATED CELLULOSE FILMS BY FUNGI

The biodegradability of Aspergillus niger (A. niger), Mucor (M-305) and Trichoderma(T-311) strains on regenerated cellulose films in media was investigated. The results showedthat T-311 strain isolated from soil adhered on the cellulose film fragments has strongerdegradation effect on the cellulose film than A. niger strain. The weights, molecular weightsand tensile strengths of the cellulose films in both shake culture and solid media decreasedwith incubation time, accompanied by producing CO_2 and saccharides. HPLC, IR and re-leased CO_2 analysis indicated that the biodegradation products of the regenerated cellulosefilms mainly contain oligosaccharides, cellobiose, glucose, arabinose, erythrose, glycerose,glycerol, ethanal, formaldehyde and organic acid, the end products were CO_2 and water.After a month, the films were completely decomposed by fungi in the media at 30℃.     

Analytical characterization of polymers used in conservation and restoration by ATR-FTIR spectroscopy

In the last few decades many new polymers have been synthesized that are now being used in cultural heritage conservation. The physical and chemical properties and the long-term behaviors of these new polymers are determined by the chemical composition of the starting materials used in their synthesis along with the nature of the substances added to facilitate their production. The practical applications of these polymers depend on their composition and form (foam, film, sheets, pressure-sensitive adhesives, heat-seal adhesives, etc.). Some materials are used in restoration works and others for the exhibition, storage and transport of works of art. In all cases, it is absolutely necessary to know their compositions. Furthermore, many different materials that are manufactured for other objectives are also used for conservation and restoration. The technical information about the materials provided by the manufacturer is usually incomplete, so it is necessary to analytically characterize such materials. FTIR spectrometry is widely used for polymer identification, and, more recently, ATR–FTIR has been shown to give excellent results. This paper reports the ATR-FTIR analysis of samples of polymeric materials used in the conservation of artworks. These samples were examined directly in the solid material without sample preparation.     

Elucidation of molecular and elementary composition of organic and inorganic substances involved in 19th century wax sculptures using an integrated analytical approach

Wax sculptures contain several materials from both organic and inorganic nature. These works of art are particularly fragile. Determining their chemical composition is thus of prime importance for their preservation. The identification of the recipes of waxy pastes used through time also provides valuable information in the field of art history.The aim of the present research was to develop a convenient analytical strategy, as non-invasive as possible, that allows to identify the wide range of materials involved in wax sculptures.A multi-step analytical methodology, based on the use of complementary techniques, either non- or micro-destructive, was elaborated. X-ray fluorescence and micro-Raman spectroscopy were used in a non-invasive way to identify inorganic pigments, opacifiers and extenders. The combination of structural and separative techniques, namely infrared spectroscopy, direct inlet electron ionisation mass spectrometry and high temperature gas chromatography, was shown to be appropriate for unravelling the precise composition of the organic substances. A micro-chemical test was also performed for the detection of starch.From this study it has been possible to elucidate the composition of the waxy pastes used by three different sculptors at the end of the 19th century. Complex and elaborated recipes, in which a large range of natural substances were combined, were highlighted.     

Evaluation of Clausena anisata essential oil from Cameroon for controlling food spoilage fungi and its potential use as an antiradical agent

Investigations were conducted to determine the chemical composition, antifungal and antiradical activities of the essential oil extracted from the fresh leaves of Clausena anisata (Willd.) Hook. F. ex Benth (from Cameroon) against Aspergillus flavus, A. niger, A. parasiticus and Fusarium moniliforme. The essential oil obtained by hydrodistillation was analysed by GC and GC/MS. The disc diffusion method was used to evaluate the fungal growth inhibition at various concentrations of the oil while the antiradical activity of the essential oil was studied by the DPPH (diphenyl picryl hydrazyl) method. The main components obtained were E-ocimenone (15.1%), Z-ocimenone (11.5%), gamma-terpinene (11.4%) and germacrene D (10.9%). After 10 days of incubation on essential oil supplemented medium, the growth of A. flavus, A. niger, A. parasiticus and F. moniliforme were totally inhibited by 4, 5, 5 and 5 mg/mL of C. anisata essential oil, respectively. The antiradical activity of C. anisata essential oil (SC50 = 5.1 g/L) was less than that of butylated hydroxyl toluene (BHT), which was used as the reference compound (SC50 = 0.007 g/L). Results obtained in the present study indicate the possibility of exploiting C. anisata essential oil to fight strains of A. flavus, A. niger, A. parasiticus and F. moniliforme responsible for biodeterioration of stored food products.     

Identification of polyimide materials using quantitative CE with UV and QTOF‐MS detection

Polyimides (PIs) are a group of widely used synthetic materials that service a variety of different purposes including microelectronics, insulating films and aerospace applications. Depending on the requirements (defined by the particular final product), the actual composition of PIs may show substantial chemical variation. To study this variation in chemical structure, CE‐MS can be employed for the determination of PI composition following chemical degradation of the polymer sample. PI is chemically decomposed to corresponding aromatic diamine and carboxylic acid components using an alkali fusion reaction. Solid polymer samples are fused in a potassium hydroxide melt yielding reaction products that are diluted in acid and can be immediately analysed by CE coupled to a Q/TOF‐MS with quantification performed using conventional UV detection. This approach involves a simple and rapid sample preparation yielding both qualitative and quantitative information regarding the chemical composition of the polymer. Application of the CE‐MS approach is shown for a range of commercially available PI and poly(amide–imide) materials and the results are used to infer the respective chemical compositions.