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 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.     

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.     

Identification of drying oils in mixtures of natural binding media used for artistic and historic works by capillary electrophoresis

To characterise the technologies and to find the appropriate treatments, the identification of the binding media of artistic and historic objects is of high importance for the restorer and conservator. Often applied together with other binders, in the present paper, the applicability of a CZE method was ascertained for the identification of the constituents of drying oils in mixtures with animal glues and/or plant gums. The drying oils are identified after alkaline hydrolysis due to their content of long‐chain saturated and unsaturated fatty acids (palmitic, stearic, oleic, linoleic, linolenic) and shorter‐chain dicarboxylic acids (pimelic, suberic, azelaic, sebacic). The dicarboxylic acids occur as products of the unsaturated acids by oxidative degradation during the drying process. The possible interferences in CE caused by the other binders are amino acids and/or monosaccharides. Such interferences could be expected as indirect UV detection has to be used, which is highly unspecific. It was found that none of the constituents of the animal glues or plant gums migrate in the time window of the analytes, thus not obstructing the identification of the analytes in any of the oil mixtures with one binder, or with a combination of both. No interference is observed even in excess of the glues. The method was applied for the identification of a drying oil in a paint layer from a ceremonial mask originating from Papua New Guinea. There is evidence that the oil is most probably from candlenut tree (Aleurites moluccana).     

Tryptic peptide analysis of protein binders in works of art by liquid chromatography-tandem mass spectrometry

A proteomics approach was used for the identification of protein binders in historical paints: the proteins were digested enzymatically into peptides using trypsin before being separated and detected by high performance liquid chromatography-electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS). Mascot (Matrix Science) was used to analyse the resulting data and for protein identification. In contrast to amino acid analysis, amino acid sequences could be studied that retain much more information about the proteins. The best extraction strategy was selected based on the number of peptides that were identified in the protein content of paint replicas using different methods. The influence of pigments on the extraction method was studied and the analytical characteristics of the selected method were determined. Finally this method was applied to historical paint microsamples on the anonymous early 15th century panel painting Crucifixion with St Catherine and St Barbara (Calvary of the Tanners), the St Catherine Altarpiece by Joes Beyaert (c. 1479) and two paintings by Pieter Brueghel the Younger (1617-1628).     

Collagen‐based proteinaceous binder‐pigment interaction study under UV ageing conditions by MALDI‐TOF‐MS and principal component analysis

This study focuses on acquiring information on the degradation process of proteinaceous binders due to ultra violet (UV) radiation and possible interactions owing to the presence of historical mineral pigments. With this aim, three different paint model samples were prepared according to medieval recipes, using rabbit glue as proteinaceus binders. One of these model samples contained only the binder, and the other two were prepared by mixing each of the pigments (cinnabar or azurite) with the binder (glue tempera model samples). The model samples were studied by applying Principal Component Analysis (PCA) to their mass spectra obtained with Matrix‐Assisted Laser Desorption/Ionization‐Time of Flight Mass Spectrometry (MALDI‐TOF‐MS). The complementary use of Fourier Transform Infrared Spectroscopy to study conformational changes of secondary structure of the proteinaceous binder is also proposed. Ageing effects on the model samples after up to 3000 h of UV irradiation were periodically analyzed by the proposed approach. PCA on MS data proved capable of identifying significant changes in the model samples, and the results suggested different aging behavior based on the pigment present. This research represents the first attempt to use this approach (PCA on MALDI‐TOF‐MS data) in the field of Cultural Heritage and demonstrates the potential benefits in the study of proteinaceous artistic materials for purposes of conservation and restoration. Copyright © 2012 John Wiley & Sons, Ltd.     

Evaluation of mass spectrometric data using principal component analysis for determination of the effects of organic lakes on protein binder identification

Matrix‐assisted laser desorption/ionisation–time of flight (MALDI‐TOF) mass spectrometry is commonly used for the identification of proteinaceous binders and their mixtures in artworks. The determination of protein binders is based on a comparison between the m/z values of tryptic peptides in the unknown sample and a reference one (egg, casein, animal glues etc.), but this method has greater potential to study changes due to ageing and the influence of organic/inorganic components on protein identification. However, it is necessary to then carry out statistical evaluation on the obtained data. Before now, it has been complicated to routinely convert the mass spectrometric data into a statistical programme, to extract and match the appropriate peaks. Only several ‘homemade’ computer programmes without user‐friendly interfaces are available for these purposes. In this paper, we would like to present our completely new, publically available, non‐commercial software, ms‐alone and multiMS‐toolbox, for principal component analyses of MALDI‐TOF MS data for R software, and their application to the study of the influence of heterogeneous matrices (organic lakes) for protein identification. Using this new software, we determined the main factors that influence the protein analyses of artificially aged model mixtures of organic lakes and fish glue, prepared according to historical recipes that were used for book illumination, using MALDI‐TOF peptide mass mapping. Copyright © 2015 John Wiley & Sons, Ltd.     

阿胶、龟甲胶中脂溶性成分的高效液相色谱指纹图谱

采用高效液相色谱法研究并建立东阿阿胶、龟甲胶脂溶性成分的指纹图谱,为药用动物胶的质量控制提供了有效的方法。采用液-液-液三相静态萃取方法制备样品,以水-乙腈为流动相进行二元梯度洗脱,检测波长为205 nm,柱温25 ℃,分析时间为60 min。采集20批样品的色谱图并对其进行相似度和聚类分析。分别标定了阿胶、龟甲胶的共有峰,其相似度分析及聚类分析结果显示两种胶间存在着明显的差异。该方法稳定可靠,可以有效地区别不同种属的药用动物胶,为动物胶剂的鉴别及质量控制提供了依据。     

Pyrolysis-gas chromatography/mass spectrometry analysis as a useful tool in forensic examination of automotive paint traces

Pyrolysis (Py)-GC/MS was applied in comparative analysis of polymer binder of 150 automobile clearcoat samples. It was found that binders of identical type and similar infrared spectra can be, in most cases, effectively differentiated, sometimes based only on the presence of peaks of very low intensity originating from minor paint components. The qualitative procedure of pyrograms comparison was developed. The results obtained, enable one to draw the conclusion that Py-GC/MS appears as valuable, very informative analytical technique of car paint samples examination. Providing identification of paint pyrolysis products, it enables discrimination of samples on the basis of their composition. However, in some cases, paint samples were still indistinguishable on the basis of their pyrograms.     

Curie-point pyrolysis gas chromatography/mass spectrometry in the identification of paint media

Curie-point pyrolysis gas chromatography/mass spectrometry has been applied to different model paint samples containing proteinaceous (egg yolk) and oily (linseed oil) media. Results of pyrolysis at 610°C are highly reproducible, and among the different pyrolysis products some compounds diagnostic for the different binders have been identified. The application of the method to real samples, i.e. from the oil painting ‘Ritratto’ by Giuseppe Nogari (1669–1763) and from the tempera paint on the ligneous roof of S. Giobbe church (16th century) in Venice, proves its effectiveness in the identification of binding media.     

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.     

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.     

Matrix-assisted laser desorption and ionisation time-of-flight mass spectrometry of Pt(II) and Pd(II) complexes

In this work, we have analysed matrix-assisted laser desorption and ionisation time-of-flight (MALDI-TOF) mass spectra of [PtCl₂(en)], [PtCl₂(dach)] and [PdCl(dien)]Cl acquired either with 2,5-dihydroxybenzoic acid (DHB) or α-cyano-hydroxycinnamic acid (CHCA) as matrices. For certain experiments, small amounts of trifluoro acetic acid (TFA) or higher concentration of inorganic salts (NaCl or KCl) was added to the matrix solution. The majority of peaks arising from the Pt(II) and Pd(II) complexes could be identified, but certain ions detectable in the spectra were generated upon ligand loss. Additionally, the analysis of Pt(II) complexes was also possible in the presence of a higher salt content, which is a commonly used analysis condition for the samples of biological origin. While DHB appears to be the best suited for MALDI-TOF mass spectrometric analysis of Pt(II) complexes, CHCA seems to be a better matrix for Pd(II) complex used in this study. On the other hand, small amounts of TFA improve the spectra quality of Pt(II) complexes, but lead most probably to the degradation of Pd(II) complex. Taken together, we have demonstrated that the analysis of metallo-drugs using MALDI-TOF MS, though accompanied with certain identification problems, is easy and reliable. On the other hand, having in mind that some complexes (i.e. a combination of a particular transition metal/ligand) cannot be analysed under conditions usually applied for others, we deem it necessary to find out the best conditions for MALDI-TOF MS analysis of each metal complex.     

Single and multiplexed immunoassays for the chemiluminescent imaging detection of animal glues in historical paint cross-sections

The characterization of the organic components in a complex, multilayered paint structure is fundamental for studying painting techniques and for authentication and restoration purposes. Proteinaceous materials, such as animal glue, are of particular importance since they are widely used as binders, adhesives and for gilding. Even though proteins are usually detected by chromatographic and proteomic techniques, immunological methods represent an alternative powerful approach to protein analysis thanks to the high specificity of antigen–antibody reactions. Our previous studies demonstrated that ovalbumin and casein could be localized in paint cross-sections with high sensitivity and good spatial resolution (i.e. within the single painting layers) by using chemiluminescent (CL) immunochemical microscope imaging. In the present research work, we describe for the first time the immunolocalization of collagen (the main protein of animal glue) in paint cross-sections by CL imaging microscopy. Two different analytical protocols have been developed, allowing either the detection of collagen or the simultaneous detection of collagen and ovalbumin in the same paint sample. The assays were used to detect collagen and ovalbumin in cross-sections from model samples and historical paintings (a wall painting dated to 1773–1774 and a painted wood panel of the Renaissance period) in order to achieve information on paint techniques and past restoration interventions.

Application of time-of-flight secondary ion mass spectrometry to automobile paint analysis.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) provides a method of elemental analysis that can distinguish among automotive paint samples of the same or nearly the same color. TOF-SIMS survey spectra were employed to determine the relative abundances of elements in the surface layers of the paint chips. The depth profile of paint samples permitted the analysis of small paint chips, the reproducible results for specific elements, and the identification of each car paint. Seventy-three samples of blue, red, white, and silver automobile paints from the major manufacturers in Korea were investigated using high resolution TOF-SIMS technique. It was found that paints of the same color produced by different manufacturers could be distinguished by this technique. TOF-SIMS is a reliable, nondestructive, and small area analyzing method for characterization of the elemental composition of automotive paint chips.     

Matrix-assisted laser desorption/ionization Fourier transform mass spectrometric analysis of oxygenated triglycerides and phosphatidylcholines in egg tempera paint dosimeters used for environmental monitoring of museum display conditions

Oxidative changes in triacylglycerols and diacylphosphatidylcholines in egg tempera paint strips are used for chemical dosimetry of the quality of the museum environment. High-resolution matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) was used as a rapid method for the determination of the exact elemental composition of the alteration products from diacylphosphatidylcholines and triacylglycerols. Light exposure of the egg tempera paints yields oxygenated diacylphosphatidylcholines and triacylglycerols. In the latter multiple incorporation of oxygen was observed as a recurring mass difference of 15.995, the exact atomic mass of oxygen. Owing to the high resolution of the FTMS data (routinely 20 000 at m/z 1000 in broadband mode), oxidation products with different elemental compositions but identical nominal mass could be distinguished. Products of oxidative cleavage of triacylglycerols were observed in samples exposed for longer times. The relative intensities of the peaks of singly and multiply oxygenated triacylglycerols were used to derive the degree of oxygenation of the egg lipids in the tempera paint dosimeters. The degree of oxygenation was found to be directly related to the light exposure time. Exposure to elevated temperature (60 degrees C) for a period of 21 days did not lead to oxygenation of the triacylglycerols and diacylphosphatidylcholines. Exposure to NO(x) and SO(2) in the dark greatly increased the degree of oxygenation. Addition of lead- or copper-containing pigments to the egg binding medium (and subsequent storage for 6 months in the dark) led to accelerated conversion of egg lipids to oxidised products.     

Characterisation of standard tempera painting layers containing proteinaceous binders by pyrolysis (/methylation)-gas chromatography-mass spectrometry

Summary Thirty standard painting layers were analysed by pyrolysis-gas chromatography/mass spectrometry (Py-GC-MS) and by Py-GC-MS in the presence of tetramethylammonium hydroxide (pyrolysis/methylation). Painting layers were prepared according to Renaissance recipes for tempera, employing proteinaceous binders (egg, glue and casein) and six different pigments. Thermal degradation products of proteins, carbohydrates and lipids were selected for semiquantitative analysis based on single/summed ion monitoring (SIM) mode. The relative distribution of these products was used to characterise binding media for the purpose of their identification in painting layers.     

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.     

Matrix-assisted laser desorption/ionization, fast atom bombardment and plasma desorption mass spectrometry of polyethylene glycol esters of (2-benzothiazolon-3-yl)acetic acid.

Fast atom bombardment (FAB), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and plasma desorption (PD) mass spectra of newly synthesized polyethylene glycols (PEGs), (M(w) 600-4000 Da) chemically modified with biologically active (2-benzothiazolon-3-yl)acetyl end-groups are described (products 1-6). The spectra were also used for the determination of the molecular mass characteristics (number average (M(n)) and weight average (M(w)) molecular masses) of the initial and modified PEGs. As expected, M(n) and M(w) of the modified samples are higher than those of the non-modified samples. However, it is shown that molecular mass dispersity (determined by the comparison of the polydispersity indices (PDI = M(w)/M(n)) of both types of PEGs) essentially do not change during this modification. The FAB mass spectra, together with molecular species, show the presence of abundant [M + Na](+) ions of product 1 and [M + Na + H](+) species of 2 and 3, and [M + Na + 2H](+) of product 4. Two main series of fragment ions, derived from the cleavage of the ether bonds, are observed. The number fractions of the molecular adduct ions and fragment adduct ions, determined from the FAB and PD mass spectra of the modified PEGs, are compared. The MALDI-TOF mass spectra of compounds 1-6 show the presence of two series of polymers. The most abundant peaks are due to [M + Na](+) and [M + K](+) ions originating from the polymers, in which the two terminal hydroxyl groups of PEGs are esterified with (2-benzothiazolon-3-yl)acetic acid. The less abundant peaks are due to the monosubstituted polymers.     

A direct and simple method of coupling matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) to thin-layer chromatography (TLC) for the analysis of phospholipids from egg yolk

Although the most important application of matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is "proteomics," there is growing evidence that this soft ionization method is also useful for phospholipid (PL) analysis. Although all PLs are detectable by MALDI-TOF MS, some lipid classes, particularly those with quaternary amines such as phosphatidylcholines (PCs), are more sensitively detected than others, and these suppress the signals of less sensitively detected PLs when complex mixtures are analyzed. Therefore, a separation of the total organic extract into individual lipid classes is necessary. As MALDI uses a solid sample, the direct evaluation of thin-layer chromatography (TLC) plates is possible. We report here on a method of directly coupling MALDI-TOF MS and TLC that can be easily implemented on commercially available MALDI-TOF devices. A total extract of hen egg yolk is used as a simple PL mixture to demonstrate the capabilities of this method. It will be shown that "clean" spectra without any major contributions from fragmentation products and matrix peaks can be obtained, and that this approach is even sensitive enough to detect the presence of PLs at levels of less than 1% of the total extract.