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.     

Classification of protein binders in artist's paints by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry: an evaluation of principal component analysis (PCA) and soft independent modelling of class analogy (SIMCA)

Proteomics techniques are increasingly applied for the identification of protein binders in historical paints. The complex nature of paint samples, with different kinds of pigments mixed into, and degradation by long term exposure to light, humidity and temperature variations, requires solid analysis and interpretation methods. In this study matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectra of tryptic-digested paint replicas are subjected to principal component analysis (PCA) and soft independent modelling of class analogy (SIMCA) in order to distinguish proteinaceous binders based on animal glues, egg white, egg yolk and milk casein from each other. The most meaningful peptide peaks for a given protein class will be determined, and if possible, annotated with their corresponding amino acid sequence. The methodology was subsequently applied on egg temperas, as well as on animal glues from different species. In the latter small differences in the MALDI-TOF mass spectra can allow the determination of a mammal or sturgeon origin of the glue. Finally, paint samples from the 16(th) century altarpiece of St Margaret of Antioch (Mlynica, Slovakia) were analysed. Several expected peaks are either present in lower abundance or completely missing in these natural aged paints, due to degradation of the paints. In spite of this mammalian glue was identified in the St Margaret samples.     

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.     

Characterization of medieval proteinaceous painting media using gas chromatography and gas chromatography — mass spectrometry

Proteinaceous organic materials used as ancient painting media were investigated by capillary gas chromatography (GC) and capillary gas chromatography — mass spectrometry (GC/MS). Medieval wall paintings made by the tempera technique were considered and their binding media were studied by the characterization of their main chemical components. The basic methodology is based on the determination of amino acids in samples of paint layers after hydrolysis and derivatization and on the comparison with reference proteinaceous materials. Multivariate chemometric techniques were used to facilitate the recognition of the protein source from chromatographic data. To characterize the binders further, a method was developed for the determination of fatty acids, present as minor components, by GC/MS. The use of fused-silica capillary columns coated with selected stationary phases allowed the separation of amino acid and fatty acid derivatives in a single analytical run.     

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.     

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.     

Analytical study of proteinaceous binding media in works of art by gas chromatography using alkyl chloroformates as derivatising agents

In this work, we present the results obtained in an analytical study of the different types of proteinaceous binding media most commonly used in paintings, using GC-FID as the technique of analysis and GC-MS as a confirmatory technique. The application of this methodology requires prior hydrolysis of the proteins in the binding media to obtain free amino acids and then volatile derivatives, in this case by reaction with chloroformates due to advantages of speed, safety and the aqueous medium in which the reaction occurs. The method proposed for the proteinaceous binding media study is to calculate the proportions of the different amino acids with respect to alanine. This method provided good characterisation of different binding media, such as pork gelatine, beef gelatine, albumin, egg white and casein. The proposed method is used for the identification of binding media (including mixtures of binders) present in real samples from paintings in the city of Valencia, Spain.     

Proteomic strategies for the identification of proteinaceous binders in paintings

The identification of proteinaceous components in paintings remains a challenging task for several reasons. In addition to the minute amount of sample available, complex and variable chemical composition of the paints themselves, possible simultaneous presence of several binders and contaminants, and degradation of the original materials due to aging and pollution are complicating factors. We proposed proteomic strategies for the identification of proteins in binders of paintings that can be adapted to overcome the requirements and difficulties presented by specific samples. In particular, we worked on (1) the development of a minimally invasive method based on the direct tryptic cleavage of the sample without protein extraction; (2) the use of microwave to enhance the enzymatic digestion yield, followed by the analysis of the peptide mixtures by nanoLC-MS/MS with electrospray ionization (ESI). Moreover, as an additional tool to tackle the problem of contaminating proteins, we exploited the possibility of generating an exclusion list of the mass signals that in a first run had been fragmented and that the mass spectrometer had to ignore for fragmentation in a subsequent run. The methods, tested on model samples, allowed the identification of milk proteins in a sample from paintings attributed to Cimabue and Giotto, thirteenth-century Italian masters, decorating the vaults of the upper church in the Basilica of St. Francis in Assisi, Italy.     

Classification of ancient proteinaceous painting media by the joint use of pattern recognition and factor analysis on GC/MS data

Reference proteinaceous binding media of the kinds historically used in painting (specially prepared for this study) and samples collected from ancient polychrome works of art were analysed by gas chromatography – mass spectrometry. The data have been processed by several multivariate chemometric techniques, such as cluster analysis, principal component analysis, factor analysis and SIMCA technique in order to classify the binding media of the ancient works of art on the basis of the proteinaceous material used. The characterisation of ancient samples was possible by the joint use of SIMCA technique and factor analysis. The latter, in particular, has made it possible to point into evidence the presence of at least two latent factors. The first factor seems to be connected with the chemical composition of the painting media, while the second factor is likely to be connected with ageing processes. Received: 14 October 1998 / Revised: 23 April 1999 / Accepted: 20 May 1999     

Classification of ancient proteinaceous painting media by the joint use of pattern recognition and factor analysis on GC/MS data

Reference proteinaceous binding media of the kinds historically used in painting (specially prepared for this study) and samples collected from ancient polychrome works of art were analysed by gas chromatography – mass spectrometry. The data have been processed by several multivariate chemometric techniques, such as cluster analysis, principal component analysis, factor analysis and SIMCA technique in order to classify the binding media of the ancient works of art on the basis of the proteinaceous material used. The characterisation of ancient samples was possible by the joint use of SIMCA technique and factor analysis. The latter, in particular, has made it possible to point into evidence the presence of at least two latent factors. The first factor seems to be connected with the chemical composition of the painting media, while the second factor is likely to be connected with ageing processes. Received: 14 October 1998 / Revised: 23 April 1999 / Accepted: 20 May 1999     

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.     

Silicone glue based solid phase microextraction fiber for amino acid determination

A solid phase microextraction fiber based on high temperature silicone glue coated on a stainless steel wire is presented for use in the extraction of amino acid derivatives. Amino acids were derivatizad using ethyl chloroformate. Effects of the extraction and desorption parameters including extraction and desorption time, desorption temperature and ionic strength on the extraction/desorption efficiency have been studied and quality parameters of the method were measured. A possibility to apply the proposed method for the identification of proteinaceous binders was demonstrated.     

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.     

Analysis of organic colouring and binding components in colour layer of art works

Two methods of analysis of organic components of colour layers of art works have been tested: IR microspectroscopy of indigo, Cu-phthalocyanine, and Prussian blue, and MALDI-TOF-MS of proteinaceous binders and a protein-containing red dye. The IR spectra distortion common for smooth outer surfaces and polished cross sections of colour layer of art works is suppressed by reflectance measurement of microtome slices. The detection limit of the three blue pigments examined is ~0.3 wt% in reference colour layers in linseed oil binder with calcite as extender and lead white as a drying agent. The sensitivity has been sufficient to identify Prussian blue in repaints on a Gothic painting. MALDI-TOF-MS has been used to identify proteinaceous binders in two historical paintings, namely isinglass (fish glue) and rabbit glue. MALDI-TOF-MS has also been proposed for identification of an insect red dye, cochineal carmine, according to its specific protein component. The enzymatic cleavage with trypsin before MALDI-TOF-MS seems to be a very gentle and specific way of dissolution of the colour layers highly polymerised due to very long aging of old, e.g. medieval, samples.     

The contribution of gas chromatography to the resynthesis of the post-Byzantine artist’s technique

Gas chromatographic analysis of ethyl chloroformate derivatives of samples taken from the paint layers of post-Byzantine panel paintings permitted the successful characterisation of the different binding media used in them. This paper describes an analytical study of various post-Byzantine binding media such as egg yolk and egg/oil emulsion, using gas chromatography. The characterisation of these icons’ binding media is an important task, as it contributes to our understanding of and the reconstruction of the post-Byzantine artists’ palette. It also enables us to investigate the validity of our assumptions about the influences of Venetian style on Greek icon painting techniques from the sixteenth to the early nineteenth century, which up to now have been based on information in artists’ handbooks. The methodology involves two experimental steps: (1) hydrolysis of the proteins and triglycerides in the binding media to obtain free amino acids and fatty acids, and (2) the formation of ethyl chloroformate derivatives via derivatization with ethyl chloroformate (ECF). This methodology is of considerable interest, since it permits the identifcation of the nature of the proteinaceous binders used in these works through the simultaneous derivatization and determination of amino acids and fatty acids. Advantages of this methodology include the small quantity of sample required and the minimum preparation time involved. The proteinaceous media can be determined based on the ratios of seven stable amino acids, while the type of emulsions and drying oils used can be determined from the fatty acid ratio. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Classification of data with missing elements and outliers

Missing elements and outliers can often occur in experimental data. The presence of outliers makes the evaluation of any least squares model parameters difficult, while the missing values influence the adequate identification of outliers. Therefore, approaches that can handle incomplete data containing outliers are highly valued. In this paper, we present the expectation-maximization robust soft independent modeling of class analogy approach (EM-S-SIMCA) based on the recently introduced spherical SIMCA method. Several important issues like the possibility of choosing the complexity of the model with the leverage correction procedure, the selection of training and test sets using methods of uniform design for incomplete data and prediction of new samples containing missing elements are discussed. The results of a comparison study showed that EM-S-SIMCA outperforms the classic expectation-maximization SIMCA method. The performance of the method was illustrated on simulated and real data sets and led to satisfactory results.     

Characterization of proteinaceous glues in old paintings by separation of the o-phtalaldehyde derivatives of their amino acids by liquid chromatography with fluorescence detection

A HPLC-fluorescence method for characterization of proteinaceous glues from binding media used in pictorial works of art prior to conservation or restoration treatment is proposed. Fluorescence derivatization of amino acids released by acid hydrolysis of standard proteins is studied. The derivatization reagent was o-phtalaldehyde with 2-mercaptoethanol as catalyst. Mobile phase was a programmed gradient among two eluents (water buffered at pH 5.8 wit 5% THF, and methanol) and is able to satisfactorily resolve the amino acid derivatives in 45 min. Peak area ratios among amino acid derivatives and the leucine derivative are useful to characterize the proteins. The method shows good sensitivity and adequate linearity between 2.0 × 10⁻³ and 3.3 mmol/l of each amino acid, with a limit of detection of 6.0 × 10⁻⁴ mmol/l. The proposed method has been successfully applied to artistic samples from items of the cultural heritage of Valencia (Spain).     

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.     

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.