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.     

Analysis of diterpenoic compounds in natural resins applied as binders in museum objects by capillary electrophoresis

The exudates of conifers consist mainly of diterpenoic acids of the abietane and pimarane type (abietic, neoabietic, dehydroabietic, palustric, pimaric, isopimaric, levopimaric and sandaracopimaric acid) and larixol acetate. These natural resins were used as adhesives, coatings, varnishes or plasticizers in artistic and historic works since ancient times. For the purpose of conservation and restoration and for art historic examination of such museum objects the identification of the binding media used is undoubtedly of paramount importance. In the present paper, the characterization of these resins based on the pattern of their diterpenoid constituents is carried out by capillary electrophoresis. For separation a background electrolyte which has been initially introduced for the analysis of chlorinated and natural resin acids in waste water was modified and the experimental conditions were adjusted in terms of resolution and analysis time. Separation was carried out in borate buffer at pH 9.25 (ionic strength 20 mmol L(-1)) with methyl-beta-cyclodextrin and sulfobutylether-beta-cyclodextrin as additives to increase selectivity and enhance the solubility of the analytes. With this electrophoretic system the resin acids of interest and larixol acetate--all as anionic cyclodextrin complexes--were separated within 5 min and detected at 200, 250 and 270 nm with a diode array detector. The electrophoretic patterns served for the characterisation of the relevant diterpenoic resins, balsams and copals. Sample pre-treatment was limited to sonication in methanol at 55 degrees C for 30 min. This enables the identification of the resins in mixtures with other binders like plant gums, animal glues or drying oils, even when these media are present in excess. Colophony was identified as resinous constituent of a modelling mass for gilded frames originating from the 19th century.     

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.     

Analysis of drying oils used as binding media for objects of art by capillary electrophoresis with indirect UV and conductivity detection

Capillary electrophoresis (CE) was applied to analyse the long-chain fatty acid composition of vegetable oils, and their degradation products formed upon ageing when drying oils are used as binding media. The analytes were detected with contactless conductivity detection (CCD) and indirect UV absorption, both detectors positioned on-line at the separation capillary. The long-chain fatty acids were resolved in a background electrolyte (BGE) consisting of phosphate buffer (pH = 6.86, 15 mM) containing 4 mM sodium dodecylbenzensulfonate, 10 mM Brij 35, 2% (v/v) 1-octanol and 45% (v/v) acetonitrile. As in this system dicarboxylic analytes, the products of oxidative degradation of unsaturated fatty acids, cannot be determined, a suitable background electrolyte was developed by the aid of computer simulation program PeakMaster. It makes use of a 10 mM salicylic acid, 20 mM histidine buffer, pH 5.85, which combines buffering ability with the optical properties obligatory for indirect UV detection. This buffer avoids system eigenpeaks, which are often impairing the separation efficiency of the system. Separation of the dicarboxylic analytes was further improved by a counter-directed electroosmotic flow (EOF), obtained by dynamically coating the capillary wall with 0.2 mM cetyltrimethylammonium bromide. Long-chain fatty acids and their decomposition products could be determined in recent and aged samples of drying oils, respectively, and in samples taken from two paintings of the 19th century.     

Characterization of bile acids and fatty acids from ox bile in oil paintings by gas chromatography-mass spectrometry

Characterization of ox bile, traditionally used in painting, is of interest in the fields of archaeometry and conservation and restoration of works of art. Bile acids, fatty acids (F), and cholesterol found in ox bile have been identified using a derivatization method that combines the formation of ethyl esters from the carboxylic groups and the trimethylsilyl ethers from hydroxyl groups. This method of analysis is consistent with these others proposed by the authors to analyze drying oils, proteins, and diterpenic resins usually used as binders and varnishes by the painters. Bile acids from binary samples such as animal glue/ox bile, casein/ox bile and Arabic gum/ox bile have been successfully analyzed using the proposed method. Finally, a method of analysis of mixtures of drying oil and ox bile has been also proposed attempting to quantitatively characterize samples in which ox bile was added to the drying oil for increasing the surfactant properties.     

Characterisation of natural polysaccharides (plant gums) used as binding media for artistic and historic works by capillary zone electrophoresis

The monosaccharide constituents of plant gums were separated by capillary electrophoresis at pH 12.1 and detected with indirect UV absorbance. The plant gums investigated were gum arabic, gum acacia, gum tragacanth, cherry gum and locust bean gum (carob gum). The monosaccharides obtained after hydrolysis with 2M trifluoroacetic acid and lyophilisation of the hydrolysate were arabinose, galactose, mannose, rhamnose, xylose, fucose, and glucose, and the two sugar acids galacturonic and glucuronic acid, in accordance with the literature. They were separated in a background electrolyte consisting of NaOH to adjust the pH, 20 mM 2,6-pyridinedicarboxylic acid as chromophore for detection and 0.5 mM cetyltrimethylammonium bromide as additive to reverse the electroosmotic flow. Based on their electropherograms, the plant gums could be identified by their typical composition (depicted in a decision scheme) as follows: a peak of glucuronic acid, together with that of rhamnose, is indicative for gum arabic. Peaks of galacturonic acid and fucose point to gum tragacanth. Locust bean gum shows a major peak for mannose (with the concomitant galactose peak in ratio 4-1), whereas a glucuronic acid and a mannose peak together with a prominent arabinose peak indicates cherry gum. The method was applied to identify the plant gums in samples like watercolours and in several paint layers like gum tempera or those with egg white or drying oils as additives. Artificial aging experiments of thin layers of gum arabic on paper or glass carried out with UV-A radiation (366 nm) did not result in changes of the saccharide patterns, in contrast to the simultaneously conducted aging of a drying oil layer.     

A study for the characterization of binding media from medieval polychrome sculptures by gas chromatography-mass spectrometry

Summary Binding media in samples taken from XIIth–XIIIth century polychrome sculptures in the Baptistery of Parma (Italy) were characterized by gas chromatography-mass spectrometry, after hydrolysis and derivatization procedures. The use of SE-52 capillary columns and the application of selected ion monitoring (SIM) allowed an increasing in the sensitivity and the ability to differentiate between animal glues, casein, egg and drying oils as components of the binders of paint and ground layers. Small amounts of material were enough for a satisfactory characterization.     

Investigation of Fatty Acid Composition of Ammi majus Seed Oil by Gas Chromatography Mass Spectrometry

The Ammi majus seeds oil constituents of methyl ester derivatives of fatty acids were analyzed using Gas Chromatography coupled to mass spectrometer. The results obtained containing the saturated as well as unsaturated fatty acids of majus seeds oils. A total of 18 different components were identified and quantified. Methyl ester of linoleic acid was found in high concentration 9.00%, among the identified analytes of interest. In addition methyl ester of Oleic acid 5.60%, Palmitic acid 3.98% Linolenic acids 1.42% were found. Concentration of the rest of identified fatty acids analytes were less than 1%. Thus from the results it is apparent that due to the presence of high percentage of valuable analytes concentrations detected in the fatty acid of A. majus, has increased its importance for the consumption in the pharmaceuticals as well as its applications in the new formulations for various skin diseases to prevent and cure from different infections.     

Identification by GC-FID and GC-MS of amino acids, fatty and bile acids in binding media used in works of art

GC-FID was used as single methodology for the identification and differentiation of proteins, lipids and ox bile from binders used in artistic paintings. The samples were hydrolyzed by HCl. Subsequently, the simultaneous formation of volatile derivatives of the amino, fatty and bile acids with ethyl chloroformate was performed quickly and safely in an aqueous medium. The derivatives were separated by capillary GC and characterized by GC-MS. The ageing of drying oils was studied, identifying pelargonic acid among other degradation products. Proteinaceous and lipoid binding media were characterized by means of the quotients between the areas of the peaks for each amino or fatty acid with respect to the area of the peak for alanine or palmitic acid. Fatty acids from ox bile were easily identified by their retention times characteristic for eicosanoic, docosanoic and pentadecanoic acids. The suggested method was applied to the analysis of binders in baroque paintings by Palomino in Valencia (Spain). Animal gelatine and linseed oil were found.     

Quantitation of triacylglycerols in plant oils using HPLC with APCI-MS, evaporative light-scattering, and UV detection

The main constituents of plant oils are complex mixtures of TGs differing in acyl chain lengths, number and positions of double bonds, and regioisomerism. A non-aqueous reversed-phase HPLC method with acetonitrile-2-propanol gradient and 30 + 15 cm NovaPak C18 columns makes possible an unambiguous identification of the highest number of TGs ever reported for these oils, based on positive-ion APCI mass spectra. A new approach to TG quantitation is based on the use of response factors with three typical detection techniques for that purpose (APCI-MS, evaporative light-scattering detection, and UV at 205 nm). Response factors of 23 single-acid TGs (saturated TGs from C7 to C22, 7 unsaturated TGs), 4 mixed-acid TGs, diolein and monoolein are calculated from their calibration curves and related to OOO. Due to differences between saturated and unsaturated acyl chains, the use of response factors significantly improves the quantitation of TGs. 133 TGs containing 22 fatty acids with 8-25 carbon atoms and 0-3 double bonds are identified and quantified in 9 plant oils (walnut, hazelnut, cashew nut, almond, poppy seed, yellow melon, mango, fig, date) using HPLC/APCI-MS with a response factor approach. Average parameters and relative fatty acid concentrations are calculated with both HPLC/APCI-MS and GC/ FID.     

Quantitative GC–MS Analysis of Artificially Aged Paints with Variable Pigment and Linseed Oil Ratios

In this study, quantitative gas chromatography–mass spectrometry (GC–MS) analysis was used to evaluate the influence of pigment concentration on the drying of oil paints. Seven sets of artificially aged self-made paints with different pigments (yellow ochre, red ochre, natural cinnabar, zinc white, Prussian blue, chrome oxide green, hematite + kaolinite) and linseed oil mixtures were analysed. In the pigment + linseed oil mixtures, linseed oil concentration varied in the range of 10 to 95 g/100 g. The results demonstrate that the commonly used palmitic acid to stearic acid ratio (P/S) to distinguish between drying oils varied in a vast range (from especially low 0.6 to a common 1.6) even though the paints contained the same linseed oil. Therefore, the P/S ratio is an unreliable parameter, and other criteria should be included for confirmation. The pigment concentration had a substantial effect on the values used to characterise the degree of drying (azelaic acid to palmitic acid ratio (A/P) and the relative content of dicarboxylic acids (∑D)). The absolute quantification showed that almost all oil paint mock-ups were influenced by pigment concentration. Therefore, pigment concentration needs to be considered as another factor when characterising oil-based paint samples based on the lipid profile.     

Identification of plant and animal glues in museum objects by GC-MS, after catalytic hydrolysis of the proteins by the use of a cation exchanger, with simultaneous separation from the carbohydrates

A method is described which enables the group-separation of proteinaceous binding media from vegetable glues (carbohydrates), and simultaneous hydrolysis of the proteins in mixtures of both. The mixtures of the binders are suspended in aqueous-ethanolic solvent with the H+ form of a strong cation exchanger and treated at elevated temperature in sealed vials. The polypeptides are cleaved by H+-catalysed hydrolysis. On abstraction the amino acids are transformed into the ammonium ions by the protons, and the cations are adsorbed by the exchanger resin. The amino acids are removed from solution in this way, thus suppressing interfering reactions with other binders, e.g. humin formation with carbohydrates. Clear and colourless solutions were obtained with all mixtures of vegetable and animal glues. Two fractions can be obtained after separation of the solid resin from the liquid supernatant - the resin fraction with the adsorbed amino acids, and the aqueous-ethanolic solution with the carbohydrates. In each of these fractions the two classes of binder can be identified separately by GC-MS; this avoids the occurrence of unresolved GC peaks and superimposed mass spectra. The method has been used to identify the binder found between fabric layers of a Burgundian liturgical vestment of the Order of the Golden Fleece from the first half of the 15th century, the Cope of the Virgin Mary. With the aid of the GC pattern obtained, and the mass spectra of the main peaks, which were identified as glucopyranose anomers, the binding medium was identified as starch.     

Identification by GC-FID and GC-MS of amino acids, fatty and bile acids in binding media used in works of art

GC-FID was used as single methodology for the identification and differentiation of proteins, lipids and ox bile from binders used in artistic paintings. The samples were hydrolyzed by HCl. Subsequently, the simultaneous formation of volatile derivatives of the amino, fatty and bile acids with ethyl chloroformate was performed quickly and safely in an aqueous medium. The derivatives were separated by capillary GC and characterized by GC-MS. The ageing of drying oils was studied, identifying pelargonic acid among other degradation products. Proteinaceous and lipoid binding media were characterized by means of the quotients between the areas of the peaks for each amino or fatty acid with respect to the area of the peak for alanine or palmitic acid. Fatty acids from ox bile were easily identified by their retention times characteristic for eicosanoic, docosanoic and pentadecanoic acids. The suggested method was applied to the analysis of binders in baroque paintings by Palomino in Valencia (Spain). Animal gelatine and linseed oil were found. Received: 27 September 2000 / Revised: 16 January 2001 / Accepted: 17 January 2001     

Gas chromatography/mass spectrometry of oils and oil binders in paintings

A GC/MS procedure has been developed, optimized, and applied to characterization of oil binders in paintings. The procedure involves hydrolysis of lipids to fatty acids (FAs) and derivatization of FAs to fatty acid methyl esters (FAMEs) by a solution of sodium methanolate in methanol at an elevated temperature. FAMEs are analyzed by temperature-programed GC followed by full-scan MS. Old and dried samples are subjected to extraction of nonpolymerized FAMEs into dichloromethane prior to hydrolysis. The method provides a good repeatability of results and has been applied to the characterization of common plant oils used in paintings, to commercial oil and tempera paints, to model painting samples, and to samples taken from real paintings. The fresh oils and binders can readily be identified and characterized. The ratio of the methyl esters of palmitic and stearic acids can be used to characterize oil binders in old works of art.     

Gas chromatographic-mass spectrometric characterisation of plant gums in samples from painted works of art

This paper presents an analytical GC-MS procedure to study the chemical composition of plant gums, determining aldoses and uronic acids in one step. The procedure is based on the silylation of aldoses and uronic acids, released from plant gums by microwave assisted hydrolysis, and previously converted into the corresponding diethyl-dithioacetals and diethyl-dithioacetal lactones. Using this method only one peak for each compound is obtained, thus providing simple and highly reproducible chromatograms. The analytical procedure was optimised using reference samples of raw plant gums (arabic, karaya, ghatti, guar, locust bean and tragacanth, cherry, plum and peach gums), commercial watercolours and paint layers prepared according to ancient recipes at the Opificio delle Pietre Dure of Florence (Italy). To identify gum media in samples of unknown composition, a decisional schema for the gum identification and the principal component analysis of the relative sugar percentage contents were employed. The procedure was used to study samples collected from wall paintings from Macedonian tombs (4th-3rd centuries bc) and from the Mycenaean "Palace of Nestor" (13th century bc) in Pylos, Greece. The presence of carbohydrates was ascertained and plant gum binders (fruit and a mixture of tragacanth and fruit tree gums) were identified in some of the samples.     

Identification of proteins,drying oils,waxes and resins in the works of art micro‐samples by chromatographic and mass spectrometric techniques

Simplified method for simultaneous identification of proteins, drying oils, waxes, and resins in the works‐of‐art samples was developed. Liquid chromatography with mass spectrometry and gas chromatography with mass spectrometry were used to identify natural materials most frequently encountered in historical paintings. Protein binders were extracted with ammonia and purified using miniaturized solid‐phase microextraction (Omix tips) to efficiently suppress matrix interferences. Zwitterionic stationary phase was used for separation of 16 underivatized amino acids analysis with hydrophilic interaction liquid chromatography that was subsequently quantified with liquid chromatography with mass spectrometry. Gas chromatography with mass spectrometry was used to analyze drying oils, waxes, and resins after one‐step saponification/transmethylation with (m‐trifluoromethylphenyl)trimethylammonium hydroxide (Meth‐Prep II). While the drawback of this reagent is low reactivity towards hydroxyl groups, sample pretreatment was much simpler as compared to the other methods. Fatty acids derivatization with the Meth‐Prep II reagent was compared with their silylation using N,O‐bis(trimethylsilyl) trifluoroacetamide/trimethylchlorosilane mixture. It was concluded that fatty acids analysis as their methyl esters instead of trimethylsilyl esters had a minor impact on the method sensitivity. The developed method was used to analyze samples from 16^th and 17^th century historical paintings.     

GC-MS分析南方红豆杉种子中的挥发油

采用水蒸气蒸馏法提取南方红豆杉种子中的挥发油，测得南方红豆杉种子挥发油的收率为2．5％。通过GC-MS技术分析了南方红豆杉种子中挥发油的化学成分。按照GC／MS通用法则，初步确定了24种化合物的结构。并用峰面积归一化法得出在挥发油中的各化学成分。其中主要成分酸类占挥发油总量的81．28％．其次的烷烃类化合物为12．74％，醛类化合物为1．80％，不饱和烷烃类化合物1．44％，醇类化合物占0、89％，酯类化合物为0．52％，胺类化合物为0．34％。这几类物质占种子中挥发油总量的99．01％。     

Identification of proteinaceous binders used in artworks by MALDI-TOF mass spectrometry

Proper identification of proteinaceous binders in artworks is essential for specification of the painting technique and thus also for selection of the restoration method; moreover, it might be helpful for the authentication of the artwork. This paper is concerned with the optimisation of analysis of the proteinaceous binders contained in the colour layers of artworks. Within this study, we worked out a method for the preparation and analysis of solid samples from artworks using tryptic cleavage and subsequent analysis of the acquired peptide mixture by matrix-assisted laser desorption/ionisation time of flight mass spectrometry. To make this approach rational and efficient, we created a database of commonly used binders (egg yolk, egg white, casein, milk, curd, whey, gelatine, and various types of animal glues); certain peaks in the mass spectra of these binders, formed by rich protein mixtures, were matched to amino acid sequences of the individual proteins that were found in the Internet database ExPASy; their cleavage was simulated by the program Mass-2.0-alpha4. The method developed was tested on model samples of ground layers prepared by an independent laboratory and then successfully applied to a real sample originating from a painting by Edvard Munch.     

Separation of highly unsaturated triacylglycerols by reversed phase HPLC with short wavelength UV detection

Reversed phase HPLC with short wavelength UV detection is a useful alternative to conventional separation systems, with RI detection, for the analysis of the triacylglycerols of highly unsaturated vegetable oils, including γ-linolenic acid-containing oils and technical drying oils. γ-Linolenic acid-containing triacylglycerols can be identified and separated from their α-linolenic analogs. The triacylglycerol fingerprints obtained by this technique from many γ-linolenic acid-containing oils and technical oils are highly characteristic, as is apparent from chromatograms obtained from the seed oils of Oenothera biennis, Borago officinalis, Ribes nigrum, Primula florindae, and Sapium sebiferum. Characteristic peak area ratios aid the identification of these oils, and estolide peaks are seen in Sapium seed kernel oils. The high detector response for triacylglycerols containing linoleate and/or linolenate residues may present additional advantages, e.g. in the detection of such triacylglycerols in olive oil.     

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.