MS Analytical Procedure for the ... - ACS Publications

Anal. Chem. 2006, 78, 4490-4500

Combined GC/MS Analytical Procedure for the Characterization of Glycerolipid, Waxy, Resinous, and Proteinaceous Materials in a Unique Paint Microsample Alessia Andreotti, Ilaria Bonaduce, Maria Perla Colombini,* Gweńae 1 lle Gautier, Francesca Modugno, and Erika Ribechini

Dipartimento di Chimica e Chimica Industriale, Universita` di Pisa, Via Risorgimento 35, 56126 Pisa, Italy

A novel GC/MS analytical procedure for the identification of lipids, waxes, proteins, and resinous materials in the same microsample from painted works of art has been optimized. It is based on a sample multistep chemical pretreatment (solvent extractions and microwave-assisted chemolysis) that is able to separate the various organic components into different fractions, which are suitably treated and derivatized before analysis. In particular, the procedure allows the complete saponification of wax esters and the completeness of the Cannizzaro type reaction of shellac acids in conditions that are suitable also for glycerides saponification. The method was tested on reference materials for the identification of proteinaceous binders (egg, collagen, casein) on the basis of the quantitative determination of the amino acid profile and the identification of glycerolipids (linseed oil, poppy seed oil, walnut oil, and egg), plant resins (Pinaceae resins, sandarac, mastic, and dammar), animal resins (shellac), tars or pitches, and natural waxes (beeswax, carnauba wax) on the basis of the determination of fatty acid, alcohol, and hydrocarbon profiles and of significant terpenic molecular markers. The procedure was applied to the characterization of three old paint microsamples. Animal glue, egg, linseed oil, beeswax, Pinaceae resin, dammar, and shellac were the identified materials found in mixtures and recognized as original and/or restoration substances. The complex chemical composition of paint layers in artworks may be related to many factors: the technique followed by the artist, the effect of aging and the environment, and the effect of past conservation practices.1 The chemical characterization of materials used in the creation and restoration of a painting is extremely useful for surveying historical events and for gaining a better knowledge of the artistic heritage. This study entails investigating the original constituent materials and those become an integral part of the work of art itself, as an effect of aging and restorations. Assessing the state of conservation of a painting is fundamental when choosing a conservative strategy based on both * Corresponding author. Tel: +390502219305. Fax: +390502219260. E-mail: [email protected]. (1) Baldini, U. Teoria del Restauro e Unita` di Metodologia; Nardini Editore: Firenze, 1978-1981.

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prevention and intervention. Developing analytical techniques for understanding the chemical composition of painting materials and studying the degradation processes is thus of paramount importance. Organic materials generally need special care and attention in the conservation of paintings, due to their relatively increased tendency with respect to the inorganic constituents to undergo degradation, transformation, and oxidation processes.2 In fact, macroscopically the paint layers may exhibit yellowing, cracking, darkening, and loss of cohesion and stability. Egg (whole, yolk, or glair), animal glue, milk or casein, drying oils (linseed, walnut, and poppy seed oils), plant resins (e.g., colophony, sandarac, mastic, dammar), animal resins (shellac), and waxes (beeswax) are the most common organic materials historically used in Europe and in the Mediterranean Basin. These materials are complex mixtures of organic species, consisting of proteins, triglycerides, terpenic compounds, sterols, hydrocarbons, esters, alcohols, free acids, etc.,3 which chemical composition and physical properties are considerably influenced by aging and environment.2,4 Organic materials in paintings may be encountered as constituents of the preparation and priming layers to reduce their porosity and water permeability, to improve the smoothness of the surface, and to avoid the penetration of fluid binding media from paint layers. For example, animal glue was commonly added to gypsum for preparation layers used in easel paintings.5 With the exception of the fresco technique in wall paintings, organic materials were used as binders to disperse pigments and to apply them on all the supports, thereby generating an adherent and elastic film. Proteinaceous materials were thus employed in the tempera technique, drying oils in the oil technique, and beeswax in encaustic painting.3,5 Easel paintings on wood and canvas were commonly covered with a transparent layer of varnish to protect the paint layer and improve color saturation. The use of natural terpenoid resins and drying oils is attested by ancient recipes for varnishes.3,5,6 A wide range of natural and synthetic organic materials was also used for restoration and consolidation.3 (2) Grattan, D. W. J. Int. Inst. Conserv.-Can. Group 1979, 4, 17-26. (3) Mills, J. S.; White, R. The Organic Chemistry of Museum Objects, 2nd ed.; Butterworth Heinemann Ltd: Oxford, 1994. (4) Sturman, L. ICCM Bull. 1980, 6, 53-60. (5) Cennini, C. Il Libro dell’Arte (XIV sec.), 1st ed.; Frezzato, F., Ed.; Neri Pozza Editore: Vicenza, 2003. 10.1021/ac0519615 CCC: $33.50

© 2006 American Chemical Society Published on Web 05/11/2006

An overview of the literature on the study and characterization of organic materials used in the production and restoration of works of art highlights how chromatographic/mass spectrometric techniques (HPLC/MS, GC/MS, Py-GC/MS) are the methods of choice for the characterization of organic species in paint samples and their behavior with aging. Above all, techniques based on GC/MS are the most commonly used. However, most works in the literature characterize single classes of organic materials, such as proteins,7-13 lipids,14-21 plant resins,22-30 animal resins,26,31 and waxes.32-35 There are only a few works on the chemical characterization and identification of more than one class of compounds in the same paint sample. Some works describe the determination of proteinaceous and glycerolipid materials based on acidic hydrolysis or methanolysis for glycerides and proteins36-40 by means of procedures based on the following: (6) Masschleiner-Kleiner, L. Ancient Binding Media, Varnishes and Adhesives, 2nd ed.; ICCROM: Roma, 1995. (7) de la Cruz-Canizares, J.; Domenech-Carbo, M. T.; Gimeno-Adelantado, J. V.; Mateo-Castro, R.; Bosch-Reig, F. J. Chromatogr. A 2004, 1025, 277285. (8) Bonaduce, I.; Colombini, M. P. Rapid Commun. Mass Spectrom. 2003, 17, 2523-2527. (9) Chiavari, G.; Lanterna, G.; Luca, C.; Matteini, M.; Prati, S.; Sandu, I. C. A. Chromatographia 2003, 57, 645-648. (10) Colombini, M. P.; Modugno, F.; Giacomelli, A. J. Chromatogr. A 1999, 846, 101-111. (11) Vallance, S. L. Analyst 1997, 122, 75R-81R. (12) Chiavari, G.; Gandini, N.; Russo, P.; Fabbri, D. Chromatographia 1998, 47, 420-426. (13) Schilling, M. S.; Khanjian, H. P. J. Am. Inst. Conserv. 1996, 35, 123-144. (14) Dron, J.; Linke, R.; Rosenberg, E.; Schreiner, M. J. Chromatogr. A 2004, 1047, 111-116. (15) Pitthard, V.; Finch, P.; Bayerova, T. J. Sep. Sci. 2004, 27, 200-208. (16) van den Berg, J. D. J.; van den Berg, K. J.; Boon, J. J. J. Chromatogr. A 2002, 950, 195-211. (17) Cappitelli, F.; Learner, T.; Chiantore, O. J. Anal. Appl. Pyrolysis 2002, 63, 339-348. (18) Languri, G. M.; van der Horst, J.; Muizebelt, W. J.; Heeren, R. M. A.; Boon, J. J. Adv. Mass Spectrom. 2001, 15, 831-832. (19) van den Berg, J. D. J.; van den Berg, K. J.; Boon, J. J. Prog. Org. Coat. 2001, 41, 143-155. (20) Gimeno-Adelantado, J. V.; Mateo-Castro, R.; Domenech-Carbo, M. T.; BoschReig, F.; Domenech-Carbo, A.; Casas-Catalan, M. J.; Osete-Cortina, L. J. Chromatogr. A 2001, 922, 385-90. (21) Chiavari, G.; Fabbri, D.; Prati, S. Chromatographia 2001, 53, 311-314. (22) Osete-Cortina, L.; Domenech-Carbo, M. T. J. Chromatogr. A 2005, 1065, 265-278. (23) Osete-Cortina, L.; Domeńech-Carbo´, M. T.; Mateo-Castro, R.; GimenoAdelantado, J. V.; Bosch-Reig, F. J. Chromatogr. A 2004, 1024, 187-194. (24) Scalarone, D.; Lazzari, M.; Chiantore, O. J. Anal. Appl. Pyrolysis 2003, 6869, 115-136. (25) Scalarone, D.; Lazzari, M.; Chiantore, O. J. Anal. Appl. Pyrolysis 2002, 64, 345-361. (26) Chiavari, G.; Fabbri, D.; Prati, S. Chromatographia 2002, 55, 611-616. (27) van den Berg, K. J.; Boon, J. J.; Pastorova, I.; Spetter, L. F. M. J. Mass Spectrom. 2000, 35, 512-533. (28) van der Doelen, G. A.; Boon, J. J. J. Photochem. Photobiol., A 2000, 134, 45-57. (29) van den Berg, K. J.; van der Horst, J.; Boon, J. J.; Shibayama, N.; de la Rie, E. R. Adv. Mass Spectrom. 1998, 14, 563-573. (30) Pastorova, I.; van der Berg, K. J.; Boon, J. J.; Verhoeven, J. W. J. Anal. Appl. Pyrolysis 1997, 43, 41-57. (31) Colombini, M. P.; Bonaduce, I.; Gautier, G. Chromatographia 2003, 58, 357-364. (32) Bonaduce, I.; Colombini, M. P. J. Chromatogr. A 2004, 1028, 297-306. (33) Regert, M.; Colinart, S.; Degrand, L.; Decavallas, O. Archaeometry 2001, 43, 549-569. (34) White, R. Stud. Conserv. 1978, 23, 57-58. (35) Regert, M.; Langlois, J.; Colinart, S. J. Chromatogr. A 2005, 1091, 124136. (36) Casoli, A.; Musini, P. C.; Palla, G. J. Chromatogr. A 1996, 731, 237246.

(i) acidic hydrolysis, derivatization of obtained free amino acids and fatty acids into N-trifluoroacetyl-0-2-propyl esters and 2-propyl esters, respectively, followed by GC/MS analysis;36 (ii) acidic methanolysis, hexane extraction, and GC/MS analysis of fatty acid methyl esters; the residual methanol fraction is then subjected to further acidic hydrolysis, derivatization of amino acids to form N-trifluoroacetyl-0-2-propyl esters, and GC/ MS analysis;37 (iii) acidic hydrolysis, chloroform extraction, derivatization with ethyl chloroformate, and GC/MS analysis of fatty acids (if there are diterpenoid and triterpenoid resins before GC/MS analysis the alcoholic moieties are silylated with trimethylsilylimidazole); the residue of the chloroform extraction, containing amino acids, is derivatized with ethyl chloroformate, extracted with chloroform, and analyzed by GC/MS.38-40 Under the above cited acidic hydrolysis conditions, a complete hydrolysis of glycerides is never achieved; fractionation of analytes between different phases is often obtained and the quantitative information are lost. Other papers41,42 report the characterization of glycerolipids, waxes and plant resins, based on hydroalcoholic saponification followed by hexane extraction, for the GC/MS analysis of neutral compounds (after derivatization with a silylating agent), and acidification and extraction with diethyl ether followed by derivatization with a silylating agent for the GC/MS analysis of the acidic compounds. Finally, an analytical procedure43,44 has been proposed for the simultaneous determination of proteins, drying oils, and plant resins based on the ammonia extraction of the sample, followed by the microwave-assisted acidic hydrolysis of the extract and the extraction of the acidic hydrolysate with diethyl ether. Drying oils are determined, after saponification of the residue of the ammonia extraction joined to the ethereal extract following the procedure described previously,41,42 and amino acids present in the acidic aqueous phase are analyzed by GC/MS after derivatization with a silylating agent. Although such procedures may give valuable information on the main compounds constituting a paint sample, none of them seems capable of determining on the same microsample the presence of drying oils, waxes, plant and animal resins, pitch or tars, and proteinaceous materials. Particularly, none of these procedures is suitable for the analysis of natural waxes and shellac. In fact, these materials require the adoption of specific hydrolysis conditions prior to GC analysis. Wax esters are particularly resistant to the hydrolysis step:32,45-47 a incomplete saponification would lead to decreased amounts of waxes acids, hydroxyacids, (37) Bersani, D.; Lottici, P. P.; Antonioli, G.; Campani, E.; Casoli, A.; Violante, C. J. Raman Spectrosc. 2004, 35, 694-703. (38) Gimeno-Adelantado, J. V.; Mateo-Castro, R.; Domenech-Carbo, M. T.; BoschReig, F.; Domenech-Carbo, A.; De la Cruz-Canizares, J.; Casas-Catalan, M. J. Talanta 2002, 56, 71-77. (39) Mateo Castro, R.; Domenech Carbo, M. T.; Peris Martinez, V.; Gimeno Adelantado, J. V.; Bosch Reig, F. J. Chromatogr. A 1997, 78, 373-381. (40) Osete-Cortina, L.; Domeńech-Carbo´, M. T.; Mateo-Castro, R.; GimenoAdelantado, J. V.; Bosch-Reig, F. J. Chromatogr. A 2004, 1024, 187-194. (41) Colombini, M. P.; Modugno, F.; Fuoco, R.; Tognazzi, A. Microchem. J. 2002, 73, 175-185. (42) Colombini, M. P.; Giachi, G.; Modugno, F.; Pallecchi, P.; Ribechini, E. Archaeometry 2003, 45, 659-674. (43) Rampazzi, L.; Cariati, F.; Tanda, G.; Colombini, M. P. J. Cult. Herit. 2002, 3, 237. (44) Rampazzi, L.; Andreotti, A.; Bonaduce, I.; Colombini, M. P.; Colombo, C.; Toniolo, L. Talanta 2004, 63, 967-977.

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alcohols, and diols, thus giving gas chromatographic profiles that do not correspond to the actual composition of the waxes. The identification of the wax could thus be compromised, especially when small quantities are present in the samples or when mixed with other lipidic materials. As far as shellac is concerned, the sesquiterpenoid acids originally constituting the resin (jalaric and laccijalaric acids) when submitted to saponification can give rise to a Cannizzaro-type reaction, leading to the formation of the corresponding acids and alcohols.31,48-53 The adoption of unsuitable hydrolysis conditions could cause a mixture of reagents and products and, thus, a decreased quantity of each detectable compound. Again, depending on the amount of the resin present in the sample, its identification could be compromised. This paper outlines a combined analytical procedure for the simultaneous characterization of proteinaceous materials, drying oils, animal and plant terpenoid resins, and natural waxes on the same microsample from painted works of art by means of GC/ MS. The procedure is based on a combination of previously published methods10,31,32,41-44 that have been modified to allow the complete saponification of wax esters32 and to allow the completeness of the Cannizzaro-type reaction of shellac acids in conditions that are suitable also for glycerides saponification.31 This procedure involves a multistep chemical pretreatment of the sample that includes acidic hydrolysis of proteins and saponification of esters. The method achieves the separation of an aqueous fraction containing amino acids and of two organic fractions (acidic and neutral), which are both submitted to derivatization reactions for silylating acidic and alcoholic polar groups and GC/MS analysis. The procedure has been tested on standard compounds, reference pure materials, and reference paint samples provided by the Superintendence of Cultural Heritage of Pisa and the Opificio delle Pietre Dure of Florence, Italy. The application of the analytical procedure to three paint samples coming from the wooden hull of a shipwreck from the 17th/18th centuries, an Italian easel painting on canvas of the same period, and an Etruscan sarcophagus from the 4th century B.C. are also reported, and the identification of organic components is discussed. EXPERIMENTAL SECTION Materials and Methods. Reagents. All the solvents were Baker HPLC grade and used without any further purification. N,Obis(trimethylsilyl)trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane and N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) with 1% trimethylchlorosilane and (45) American Society for Testing and Materials. Standard Test Method for Saponification Number (Empirical) of Synthetic and Natural Waxes. ASTM Method D1387-89: ASTM Philadelphia, PA, 1989. (46) Jimenez, J. J.; Bernal, J. L.; Aumente, S.; Toribio, L.; Bernal, J., Jr. J. Chromatogr. A 2003, 1007, 101-116. (47) Evershed, R. P.; Dudd, S. N. J. Archaeol. Sci. 2003, 30, 1-12. (48) Singh, A. N.; Upadhye, A. B.; Mhaskar, V. V.; Sukh, D. Tetrahedron 1974, 30, 867-874. (49) Khurana, R. G.; Singh, A. N.; Upadhye, A. B.; Mhaskar, V. V.; Sukh, D. Tetrahedron 1970, 26, 4167-4175. (50) Wadia, M. S., Khurana, R. G.; Mhaskar, V. V.; Sukh, D. Tetrahedron 1969, 25, 3841-3854. (51) Singh, A. N.; Upadhye, A. B.; Wadia, M. S.; Mhaskar, V. V.; Sukh, D. Tetrahedron 1969, 25, 3855-3867. (52) Upadhye, A. B.; Wadia, M. S.; Mhaskar, V. V.; Sukh, D. Tetrahedron 1970, 26, 4177-4187. (53) Upadhye, A. B.; Wadia, M. S.; Mhaskar, V. V.; Sukh, D. Tetrahedron 1970, 26, 4387-4396.

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triethylamine were purchased from Sigma-Aldrich (USA). The following solutions, apart from those for the amino acids, were prepared by weighing pure substances and were used as standards: (i) amino acids solution in 0.1 N HCl, purchased from SigmaAldrich (USA) and containing 12.5 µmol/mL of proline (Pro) and hydroxyproline (Hyp) and 2.5 µmol/mL of aspartic acid (Asp), glutamic acid (Glu), alanine (Ala), arginine, cysteine, phenylalanine (Phe), glycine (Gly), hydroxylysine, isoleucine (Ile), histidine, leucine (Leu), lysine (Lys), methionine (Met), serine (Ser), tyrosine (Tyr), threonine, and valine (Val); (ii) fatty acids solution in acetone, containing lauric acid (0.24 mg/g), suberic acid (0.27 mg/g of Su), azelaic acid (0.28 mg/g of A), myristic acid (0.25 mg/g of My), sebacic acid (0.3 mg/g of Se), palmitic acid (0.25 mg/g of P), oleic acid (0.51 mg/g of O), stearic acid (0.51 mg/g of S). All acids, purity g 99%, were purchased from Sigma-Aldrich (USA); (iii) terpenoid acids solution in acetone containing abietic acid (0.78 mg/g, Sigma-Aldrich (USA), purity 70%) dehydroabietic acid (0.32 mg/g, Sigma-Aldrich (USA), purity 30%), oleanolic acid (1.14 mg/g, Extrasyntese (France), purity 99%), and ursolic acid (1.08 mg/g, Extrasyntese (France), purity 99%); (iv) neutral compounds solution in hexane containing dipterocarpol (0.89 mg/g, Sigma-Aldrich (USA), purity 99%), 1-dodecanol (1.05 mg/g, Fluka (USA), purity 99%), R-amyrine (0.96 mg/g, Extrasynthese (France), purity 99%), and cholesterol (0.83 mg/ g, Sigma-Aldrich (USA), purity 99%); (v) aleuritic acid (Fluka (USA), purity 95%) solution in ethanol (1.01 mg/g); (vi) tripalmitine (Sigma-Aldrich (USA), purity 99%) solution in hexane (1.24 mg/g); (vii) norleucine solution in bidistilled water (Sigma-Aldrich (USA), purity 99%), 138.66 µg/g, was used as derivatization internal standard for amino acids; (viii) tridecanoic acid solution of isooctane (Sigma-Aldrich (USA), purity 99%), 135.48 µg/g, was used as acidic and neutral fraction derivatization internal standard; (ix) hexadecane solution in isooctane (Sigma-Aldrich (USA), purity 99%), 80.34µg/g, was used as injection internal standard. All standard solutions were used to derive calibration curves. Apparatus. Microwave oven model MLS-1200 MEGA Milestone (FKV, Sorisole (BG,) Italy), 6890N GC system gas chromatograph (Agilent Technologies) coupled with a 5973 mass selective detector (Agilent Technologies) single quadrupole mass spectrometer equipped with PTV injector were used. The mass spectrometer was operated in the EI positive mode (70 eV). The MS transfer line temperature was 280 °C; the MS ion source temperature was kept at 230 °C; and the MS quadrupole temperature was at 150 °C. The mass spectrometer operated in the EI positive mode (70 eV). For the gas chromatographic separation, an HP-5MS fused silica capillary column (5% diphenyl/95% dimethyl-polysiloxane, 30 m × 0.25 mm i.d., 0.25 µm film thickness, J&W Scientific, Agilent Technologies, Palo Alto, CA) with a deactivated silica precolumn (2 m × 0.32 mm i.d., J&W Scientific Agilent Technologies, Palo Alto, CA) was used. The carrier gas was used in the constant flow mode (He, purity 99.995%) at 1.2 mL/min.

For fatty acids, terpenoid acids and neutral compounds, alcohols, and hydrocarbons analysis the PTV injector was used in splitless mode at 300 °C. The chromatographic oven was programmed as follows: 80 °C, isothermal for 2 min, 10 °C/min up to 200 °C, 200 °C, isothermal for 3 min, 10 °C/min up to 280 °C, 280 °C, isothermal for 3 min, 20 °C/min up to 300 °C, 300 °C, isothermal for 20 min. For the amino acids analysis, the PTV injector was used in splitless mode at 220 °C. The chromatographic oven was programmed as follows: initial temperature 100 °C, isothermal for 2 min, then 4 °C/min up to 280 °C, and isothermal for 15 min. MS spectra were recorded both in TIC (total ion current) and SIM (single ion monitoring) mode. Mass Spectra Assignment. Mass spectral assignment was based on the direct match with the spectra of Wiley 275 library, and comparisons with mass spectra of pure compounds, when available, were made. In the absence of reference spectra, the peak assignment was based on mass spectra interpretation. Samples. Reference Materials. (a) Glycerolipids. Layers of linseed oil, walnut oil, and egg applied on the glue/gypsum preparation of cherry tree wood tiles were prepared by restorers at the Cultural Heritage offices in Pisa in 1995 and stored in the dark at room conditions. Samples in the range of 0.5-1 mg were analyzed. (b) Plant Resins. Layers of colophony (C9) on a glass support were prepared at the laboratory of the Opificio delle Pietre Dure (Florence) in 1976 and naturally aged at room conditions. Layers of sandarac (Sn-v), mastic (Ms-v), and dammar (Dm-v) on glass support belong to a collection of reference paint layers prepared in 1996 at the Opificio delle Pietre Dure. In 1998, they were subjected to artificial aging according to the following program for 4 weeks: Monday, Wednesday, and Friday: NOx 10 ppm, SO2 10 ppm, 30 °C, RH 75%, 24 h; Tuesday, Thursday, Saturday, and Sunday: UV (365 nm) 30 °C, RH 75%, 24 h.54 They were stored at room conditions. Samples in the range of 0.2-0.5 mg were analyzed. (c) Beeswax. Pure beeswax from Apis mellifera was purchased from a local apiarist. Samples in the range of 0.5-1 mg were analyzed. (d) Carnauba Wax. Carnauba wax was supplied by Fluka (USA). Samples in the range of 0.5-1 mg were analyzed. (e) Shellac. Pure shellac (“Lacca in Lacrime” coming from East India), naturally aged from the early 19th century, belonging to the Salvemini Collection was kindly provided by the Polo Tecnico Statale, Florence, Italy. Samples in the range of 0.2-0.5 mg were analyzed. Paint Samples from Artworks. (a) Sample A: Painted Hull of a Shipwreck. The wreck, named Dor 2002/2, was found in the Tantura lagoon, an underwater excavation located near the Mediterranean Israeli coasts. It has been dated back to the late 1700s/early 1800s. The wreck was excavated by a combined expedition to the Leon Recanati Institute for Maritime Studies at the University of Haifa (Dr. Yaacov Kahanov and Ms. Deborah Cvikel), the Nautical Archaeology Society of Great Britain, and the Aqua Dora Diving Centre. (54) Lanterna, G.; Mairani, A.; Mattini, M.; Rizzi, M.; Vigato, A. Proceedings of the 2nd International Congress on Science and Technology for the Safeguard of Cultural Heritage in the Mediterranean Basin, Paris, July 5-9, Consiglio Nazionale delle Ricerche, Rome, 1999.

Figure 1. Scheme of the GC/MS combined analytical procedure.

While excavation is still in progress, a chemical study has been started regarding the technology and the materials used in the ship’s painting. The sample weight was 0.2 mg. (b) Sample B: Easel Painting. The painting on canvas, Baptism of Christ with St Lucy and St Apollonia, is located in the Santa Maria Santissima Church in Sorgnano (Carrara, Italy) and was produced in the late 1600s/early 1700s. The painter is unknown. The painting is currently being restored, and an analytical study has been undertaken to choose the most suitable conservation practice. A sample (0.4 mg) was taken from the white painted area above Christ’s head. (c) Sample C: A Painted Etrurian Sarcophagus. The Sarcofago delle Amazzoni was found in Tarquinia, an ancient Etrurian city close to Rome, in 1869 by G. Bruschi and is now conserved at the Archaeological Museum in Florence, Italy. It is constituted by a calcareous alabaster case painted on all sides. The painting is presumed to be from the 4th century B.C. Preliminary analytical studies have been made on the painting technique and on identifying the presence of materials ascribable to past restorations, which are not documented. The sample C (0.9 mg; SA17) was taken from the coat of a gray horse.55 Analytical Procedure. The analytical procedure is reported in Figure 1. The various steps are as follows: I. The sample is subjected to ammonia extraction. To solubilize proteins and to separate the proteinaceous matter from insoluble inorganic salts, such as calcium carbonate, that can interfere in amino acid analysis,10 300-400 µL of 2,5 N NH3 are added to the sample in an ultrasonic bath at 60 °C for 120 min, twice. During this step, free acids with a certain solubility in ammonia are extracted together with the proteinaceous matter. The residue containing insoluble organic and inorganic species is kept for step VI. II. The extracted ammonia solution (containing proteinaceous matter10,43) is evaporated to dryness under a stream of nitrogen and then subjected to acidic hydrolysis assisted by microwave (power ) 250 W for 10 min; power ) 500 W for 30 min) in the vapor phase with 30 mL of 6 N HCl at 160 °C for 40 min. In these experimental conditions, it is possible to find a good compromise (55) Bonaduce, I.; Bottini, A.; Colombini, M. P.; Giachi, G.; Modugno, F.; Pallecchi, P. Sci. Technol. Cult. Herit. 2004, 13, 89-96.


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between the effectiveness of the hydrolysis of the more stable peptide bonds (Ile-Ile, Val-Val, Ala-Ala, Ile-Ala) and the need to reduce the losses of the more chemically labile amino acids. III. After the hydrolysis, bidistilled water (200-400 µL) is added to the acidic hydrolysate, which is then extracted with diethyl ether (200 µL, three times). Free fatty acids and other organic compounds extracted together with proteins by ammonia in step I are solubilized in ether. The ethereal extracts combined with the residue of the ammonia extraction derived from step I are kept for step VI. The aqueous phase after the diethyl ether extraction is an acidic solution containing amino acids: amino acid fraction. IV. An aliquot of the acid amino acid fraction is evaporated to dryness under a stream of nitrogen (repeated twice by adding a few drops of acetone) and is subjected to derivatization with 10 µL of N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide) (MTBSTFA), 40 µL of pyridine (solvent), 2 µL of triethylamine (catalyst), and 5 µL of norleucine solution at 60 °C for 30 min and then added with 5 µL of hexadecane solution. V. At total of 2 µL of the pyridine solution of derivatized amino acids is analyzed by GC/MS. VI. The residue of the ammonia extraction, combined with the ethereal extract of the hydrolyzed solution, is subjected to saponification assisted by microwaves (power ) 200 W) with 300 µL of KOHETOH 10% wt at 80 °C for 60 min. The use of microwaves allows saponification not only of glycerides but also of wax esters, which are particularly resistant to hydrolysis in conventional conditions32 and to obtain the completeness of the Cannizzarotype reactions of shellac terpenoid acids.31 VII. After saponification, the hydroalcoholic solution is diluted in bidistilled water, and the unsaponifiable is extracted in hexane (200 µL, three times). The extract contains neutral organic molecules: neutral fraction. The residue is kept for step X. VIII. An aliquot of this extract is evaporated to dryness under nitrogen stream and subjected to derivatization with 20 µL of N,Obistrimethylsilyltrifluoroacetamide (BSTFA), 200 µL of isooctane (solvent), and 5 µL of tridecanoic acid solution at 60 °C for 30 min. Finally 5 µL of hexadecane solution is added. IX. A total of 2 µL of the isooctane solution of neutral compounds, eventually derivatized, is analyzed by GC/MS. The analysis of the neutral fraction allows the neutral terpenoid compounds, sterols, alcohols, and alkanes to be determined. X. The residue of the n-hexane extraction is acidified with trifluoroacetic acid (aqueous solution 1:1) and then extracted with diethyl ether (200 µL, three times). The use of trifluoroacetic acid allows the solubilization of aleuritic acid, one of the main components of shellac.31 The extract containing organic acids is dried under nitrogen flow (to remove trifluoroacetic acid partially soluble in the organic phase) and redissolved in acetone: acidic fraction. XI. An aliquot of the acidic fraction is then subjected to the same procedure for derivatization described for the neutral fraction. XII. A total of 2 µL of the isooctane solution of derivatized acidic compounds is analyzed by GC/MS. The analysis of the acidic fraction allows the determination of monocarboxyic acids, dicarboxylic acids, hydroxy acids, and terpenoid acids. 4494 Analytical Chemistry, Vol. 78, No. 13, July 1, 2006

Table 1. Detection and Quantitation Limits of Fatty Acids, Dicaboxylic Acids, and Amino Acid Standard Compounds compound lauric acid, suberic acid, myristic acid, sebacic acid, oleic acid azelaic palmitic and stearic acid Met, Hyp, Tyr Ala, Gly, Val, Ile, Ser, Phe, Asp, Lys Leu, Prot, Glu

detection limit (µg)

quantitation limit (µg)

0.1-0.3

0.3

0.3 0.6 1.0 0.01-0.03 0.04-0.06 0.08-0.18

0.5 1.6 2.5 0.01-0.04 0.1 0.2

RESULTS AND DISCUSSION The combined analytical procedure was tested by using standard solutions and reference materials. Running blanks of the procedure highlighted a low level of contamination of amino acids and fatty acids. As a result, the detection limit and the quantitation limit were calculated on the basis of the calibration curves as the analyte concentration, giving a chromatographic peak whose area was given by the sum of the mean blank signal plus 3 and 10 times the blank standard deviation, respectively. The results for fatty acids, dicaboxylic acids, and amino acidic standard compounds are reported in Table 1. Abietic acid, dehydroabietic acid, R-amyrine, dipterocarpol, cholesterol, ursolic acid, oleanolic acid, 1-dodecanol, and aleuritic acid gave no signals in the blanks. The detection limits were calculated and were in the range of 1-4 ng. The low amounts of analytes that is possible to determine indicate that the combined analytical procedure is suitable for analyzing samples taken from painted artworks, which contain often only a minor fraction of organic material56 and are actually in the size of hundreds of micrograms. Finally, to test the behavior of glycerolipids in the saponification step assisted by microwaves, tripalmitine was subjected to the analytical procedure: a saponification yield of 93% with a relative standard deviation of 12% was obtained on five replicate experiments. Analysis of Reference Materials. Proteinaceous Materials. As far as the proteinaceous material is concerned, the procedure does not differ from that already published.10,44 In any case, using the reference paint samples containing animal glue and egg, the absence of amino acids in the other extracted phases was assessed. The identification of the proteinaceous material (egg, casein, or animal glue) in unknown samples can be performed by principal component analysis (PCA) of amino acidic percentage content data, using a reference data set of 101 reference samples containing egg, casein, and animal glue10 belonging to the paint reference collection of the Opificio delle Pietre Dure, Florence.54 In particular, the PCA is performed, using XLSTAT 6.0 (Addinsoft, France), on the correlation matrix of the data. The first two components account for 95,3% of the variance of the data. Glycerolipids. The quantitative determination of the percentage content of five monocarboxylic fatty acids (lauric, miristic, palmitic, oleic, and stearic acids) and of three dicarboxylic acids (suberic, azelaic, and sebacic acids) allows us to distinguish between (56) Colombini, M. P.; Modugno, F. J. Sep. Sci. 2004, 27, 147-160.

Table 2. Average Values of the Characteristic Parameters Calculated for the Reference Paint Samples

A/P P/S ΣD cholesterol

linseed oil

walnut oil

egg

1.3 ( 0.2 1.2 ( 0.2 42.8 ( 3.5

1.4 ( 0.2 2.2 ( 0.3 38.2 ( 4.1

0.2 ( 0.1 2.4 ( 0.3 11.9 ( 1.8 yes

different triglycerides sources (drying oil or egg) and to determine the kind of drying oil (poppyseed, walnut, linseed) on the basis of characteristic parameters: A/P (azelaic over palmitic acid ratio), P/S (palmitic over stearic acid ratio), ΣD (sum of dicarboxylic acids).41 The characteristic parameters in the reference layer samples were evaluated. The mean results on triplicate analyses are reported in Table 2 and agree with the literature values.41 It has to be underscored that these parameters represent reference values for pure materials: they must be carefully evaluated whenever the analytical results suggest the simultaneous occurrence of more than one source of lipid materials. Beeswax. In the acidic fraction, linear monocarboxylic acids with an even number of carbons (ranging from 12 to 34 atoms and showing a maximum with palmitic acid) and linear ω-1 hydroxy acids with an even number of carbons (ranging from 16 to 26 atoms and showing a maximum with 15-hydroxyhexadecanoic acid) were identified. In the neutral fraction linear hydrocarbons with an odd number of carbons (ranging from 23 to 33 and showing a maximum with eptacosane), linear alcohols with an even number of carbons (ranging from 24 to 34 and showing a maximum with triacontanol), and linear R-(ω-1) diols with an even number of carbons (ranging from 22 to 34) were identified. The chromatographic profiles are in agreement with the literature, and the absence of wax esters, or at least, their presence below their detection limit, shows how efficient saponification is.32 Since palmitic acid is the most abundant compound in the hydrolyzed wax, representing about 15% w/w,57 the estimation of the saponification yield was related to its recovery. By using the suggested procedure, a palmitic acid weight percentage content of 20.1% with a relative standard deviation of 6% calculated on five replicates was obtained. This result is in good agreement with the literature data57 and is much higher in respect to that obtained applying the procedure used for the characterization of plant resins and oils in artistic objects.42 In fact, by using this last procedure, which employs KOHMEOH 10%:KOHH2O 10% (1:1) for 3 h at 60 °C in a water bath, a palmitic acid weight percentage content of 3.2% with a relative standard deviation of 14% calculated on five replicates was obtained.32 Carnauba Wax. In the acidic fraction (Figure 2 and peak assignment in Table 3), linear monocarboxylic acids with an even number of carbons (ranging from 16 to 32 atoms and showing a maximum with tetracosanoic acid) and linear ω hydroxy acids with an even number of carbons (ranging from 16 to 30 atoms and showing a maximum with 28-hydroxyoctacosanoic acid), 3-hydroxycinnamic acid, 4-methoxycinnamic acid, and 4-hydroxycinnamic acid were identified. (57) Downing, T.; Krantz, Z. H.; Lamberton, J. A.; Murray, K. E. Aust. J. Chem. 1961, 14, 251-263.

Figure 2. Total ion chromatogram of the carnauba wax acidic fraction. The identified compounds are listed in Table 3. Table 3. Compounds Identified in the Carnauba Wax Acidic Fractiona peak no.

compound

peak no.

compound

1 2 3 4 5 6 7 8 9 10 11

3-hydroxycinnamic acid 4-methoxycinnamic acid 4- hydroxycinnamic acid 3,4-dihydroxycinnamic acid palmitic acid oleic acid stearic acid 16-hydroxyhexadecanoic acid eicosanoic acid 18-hydroxyoctadecanoic acid docosanoic acid

12 13 14 15 16 17 18 19 20 21 22

20-hydroxyeicosanoic acid tetracosanoic acid 22-hydroxydocosanoic acid hexacosanoic acid 24-hydroxytetracosanoic acid octacosanoic acid 26-hydroxyhexacodanoic acid triacontanoic acid 28-hydroxyoctacosanoic acid dotriacontanoic acid 30-hydroxytriacontanoic acid

a Chromatogram shown in Figure 2 (alcoholic and acidic compounds are present as TMS derivatives).

Figure 3. Total ion chromatogram of the carnauba wax neutral fraction. The identified compounds are listed in Table 4. Table 4. Compounds Identified in the Carnauba Wax Neutral Fractiona peak no.

compound

peak no.

compound

23 24 25 26 27 28 29

docosanol tetracosanol 1,22-docosandiol hexacosanol 1,24-tetracosandiol octacosanol 1,26-hexacosandiol

30 31 32 33 34 35 36

triacontanol 1,28-octacosandiol dotriacontanol 1,30-triacontandiol tetratriacontanol 1,32-dotriacontandiol 1,34-tetratriacontandiol


In the neutral fraction (Figure 3 and peak assignment in Table 4), linear alcohols with an even number of carbons (ranging from 22 to 34 and showing a maximum with dotriacontanol) and linear R-ω diols with an even number of carbons (ranging from 22 to 34) were identified. Analytical Chemistry, Vol. 78, No. 13, July 1, 2006

4495

Table 5. Compounds Identified in the Dammar Acid Fraction and the Neutral Fractiona peak no.

compound

37 38 39

shoreic acid 20,24-epoxy-25-hydroxydammaren-3-one 20,24-epoxy-25-hydroxydammaren-3-ol

a Chromatograms shown in Figure 4, panels a and b, respectively (alcoholic and acidic compounds are present as TMS derivatives).

Figure 4. (a) Total ion chromatogram of dammar resin acid fraction. (b) Total ion chromatogram of dammar resin neutral fraction. The identified compounds are listed in Table 5.

The chromatographic profiles are in agreement with the literature,58 and the absence of carnauba wax esters, or at least, their presence below the detection limit, again underscores the efficacy of the saponification step assisted by microwaves. Moreover, since tetracosanoic acid is the most abundant compound in the hydrolyzed wax,58 the estimation of the saponification yield was related to its recovery. The tetracosanoic acid weight percentage content, calculated on five replicates, was 30.0% with a relative standard deviation of 6%. This result was compared to that obtained with the previously cited analytical procedure proposed for the simultaneous determination of plant resins and lipids42 showing a much higher saponification yield: 4.3% was the tetracosanoic acid weight percentage content obtained, with a relative standard deviation of 8% on five replicates. Plant Terpenoid Resins. In the acidic fraction of the colophony sample, the following compounds were identified: palustric, pimaric, sandaracopimaric, didehydroabietic, dehydroabietic 7-oxodehydroabietic, 7-oxo-didehydroabietic, and 15-hydroxy-7-oxodehydroabietic acids. The chromatographic profile was in accordance with the literature,59 and the presence of palustric acid indicates that abietane skeletons have not yet undergone a complete aromatization. In the acidic fraction of the sandarac sample, pimaric, sandaracopimaric, and isopimaric acids, turosolic and agatic acids, and totarolon were identified, with a chromatographic profile in accordance with the literature.24 With regard to the dammar sample, the chromatogram of acidic and neutral fractions are reported in Figure 4, and the identified compounds are listed in Table 5. Shoreic (eichlerianic) acid in the acidic fraction and 20,24epoxy-25-hydroxydammaren-3-one and 20,24-epoxy-25-hydroxy(58) Downing, T.; Krantz, Z. H.; Murray, K. E. Aust. J. Chem. 1961, 41, 619627. (59) Colombini, M. P.; Modugno, F.; Ribechini, E. J. Mass Spectrom. 2005, 40, 675-687.


Figure 5. (a) Total ion chromatogram of the mastic acid fraction. (b) Extracted ion chromatogram of ion 143 of the mastic neutral fraction. Identified compounds are reported in Table 6.

dammaren-3-ol in the neutral fraction were detected. The two last compounds are formed by oxidation of the side chain of dammarane molecules and are known to be stable aging products present in dammar-based varnish.28,60,61 Finally, the chromatogram of acidic and neutral fraction of the mastic sample are reported in Figure 5, and the compounds identified are listed in Table 6. The presence of 2,4-sec-28-norolean-12-en-3,28-dioic, 2,4-sec-28norolean-18-en-3,28-dioic, moronic, oleanonic, and masticadienoic acids in the acidic fraction and 20,24-epoxy-25-hydroxydammaren3-one in the neutral fraction were found. This sample shows the occurrence of stable aging and oxidation products that are not present in the native resin but have been recognized in aged mastic resin varnishes (2,4-sec-28-norolean-12-en-3,28-dioic acid and 2,4-sec-28-norolean-18-en-3,28-dioic acid).62 Shellac. In the acidic fraction, the presence of butolic, epilaccilaksholic, epilaccishellolic and laccishellolic, epilaksholic and laksholic, epishellolic and shellolic, and 16-hydroxyhyxadecan-9enoic acids; aleuritic acid and its derivative; and traces of jalaric (60) Boon, J. J.; van der Doelen, G. A. Postprints of the International colloquium: Firnis; Material Aesthetik Geschichte: Braunschweig, 1998; pp 92-104. (61) Colombini, M. P.; Modugno, F.; Giannarelli, S.; Fuoco, R.; Matteini, M. Microchem. J. 2000, 67, 385-396. (62) Scalarone, D. Caratterizzazione e studi di invecchiamento di materiali pittorici organici naturali e sintetici, Ph.D. Thesis, 2001.

Table 6. Compounds Identified in the Mastic Resin Acidic Fraction and the Neutral Fractiona peak no.

compound

40 41 42 43 44 38

3,4-sec-28-norlean-12-en-3,28-dioic acid 3,4-sec-28-norlean-18-en-3,28-dioic acid moronic acid oleanonic acid masticadienoic acid 20,24-epoxy-25-hydroxydammaren-3-one

a

Chromatograms shown in Figure 5, panels a and b, respectively (alcoholic and acidic compounds are present as TMS derivatives).

Figure 7. Total ion chromatogram of the neutral fraction of sample A. The identified compounds are listed in Table 8. Table 8. Compounds Identified in Sample A Neutral Fractiona

Figure 6. Total ion chromatogram of the acid fraction of sample A. The identified compounds are listed in Table 7.

a

peak no.

compound

54 55 56 57 58 59 60 61

19-norabieta-8,11,13-triene 19-norabieta-4,8,11,13-tetraene 18-norabieta-8,11,13-triene tetrahydroretene retene simonellite C19H20, higher retene homolougue methyl-dehydroabietate

Chromatogram shown in Figure 7.

Table 7. Compounds Identified in Sample A Acidic Fractiona peak no.

compound

peak no.

compound

45 46 47 48 49 5

lauric acid suberic acid azelaic acid myristic acid sebacic acid palmitic acid

6 7 50 51 52 53

oleic acid stearic acid di-dehydroabietic acid dehydroabietic acid abietic acid 7-oxo-dehydroabietic acid


and laccijalaric acids31 were highlighted. The chromatographic profile showed that the Cannizzaro-type reaction was almost complete.31 Finally, it has to be highlighted that the amino acid content of the drying oils, waxes, and plant and animal resins was at the blank level, and no other significant compounds were detected in the amino acidic fraction. Analysis of Paint Samples from Artworks. Painting on the Hull of a Shipwreck, Late 1700s/Early 1800s, Sample A. The amino acid analysis showed a non significant content of these compounds, thus suggesting the absence of proteinaceous materials. Figure 6 shows the chromatogram of the acidic fraction of the paint sample, and Table 7 reports the identified compounds. On the basis of quantitative analysis of fatty acids and of dicarboxylic acids, the following parameters were found: A/P ) 1.4, P/S ) 1.1, and ΣD ) 45.9%. These values, with A/P ratio >1 and the high content of dicarboxylic acids suggest that a drying oil was used as a binder. According to the P/S value, the use of linseed oil is suggested. The occurrence of diterpenoid acids with abietane skeletons, namely, dehydroabietic, di-dehydroabietic, and 7-oxo-dehydroabietic acids, is indicative of a resin obtained from trees of the Pinaceae family. However, the presence of neutral

aromatized diterpenoid molecules in the neutral fraction (Figure 7, Table 8) and, in particular, retene and norabietanes, sustains the occurrence of tarry or pitchy substances. Retene and norabietanes are, in fact, the final products of dehydrogenation, demethylation, and decarboxylation reactions that occur during the thermal treatment required to produce tar and pitch from resin.42,59,63,64 Moreover, the presence of methyldehydroabietate and methyl-abietate suggests that the pitch was obtained by pyrolytic treatments of resinous wood from Pinaceae plants. In fact, during hard heating of wood, high amounts of gaseous methanol are produced and can easily react with diterpenoid acids to form the corresponding esters.42,63,64 These results highlight the use of linseed oil as a binder for the painted ship 2002/2. The presence of tarry like material may be due to the waterproofing material present on the wood underneath the paint layer. Actually, resinous materials, waxes and tar or pitch were commonly used for finishing the wood of ships,42,63,64,65 thus a painting technique based on a mixture of pitch and drying oil seems extremely improbable. Easel Painting, 18th-Beginning of 19th Centuries, Sample B. The analysis of the amino acid fraction highlighted the presence of proteinaceous material. The amino acids percentage content is reported in Table 9. The occurrence of hydroxyproline, a marker of collagen, indicates that animal glue is present. The PCA analysis, whose score plot is shown Figure 8, does not assign the sample to any cluster. Anyway, due to the position of the sample between the animal glue and the egg clusters, the hypothesis of the occurrence of egg together to animal glue seems reasonable. (63) Pollard, A. M.; Heron, C. Archaeological Chemistry; RSC Paperbacks: Cambridge, 1996; pp 239-270. (64) Robinson, N.; Evershed, R. P.; Higgs, W. J.; Jerman, K.; Eglinton, G. Analyst 1987, 112, 637-643. (65) Connan, J.; Nissenbaum, A. J. Archaeol. Sci. 2003, 30, 709-719.


4497

Table 9. Amino Acidic Relative Percentage Content of Samples B and C sample

Ala

Gly

Val

Leu

Ile

Met

Ser

Pro

Phe

Asp

Glu

Lys

Hyp

Tyr

B C

6.7 9.9

19.0 20.1

3.2 6.8

5.6 5.4

2.4 4.6

0.0 0.0

8.9 7.0

2.8 14.8

4.4 4.1

12.8 13.3

14.4 12.6

0.1 0.0

7.1 1.3

2.7 0.0

Table 10. Compounds Identified in Sample B Acid Fractiona

Figure 8. PCA score plot relative to samples B and C. The relative amino acid percentage content of the two samples is reported in Table 9.

peak no.

compound

peak no.

compound

45 46 47 48 49 62 5 6 7 63 64 50 51

lauric acid suberic acid azelaic acid miristic acid sebacic acid undecandioic acid palmitic acid oleic acid stearic acid 14-hydroxyhexadecanoic acid 15-hydroxyhexadecanoic acid di-dehydroabietic acid dehydroabietic acid

9 65 53 11 13 15 37 43 66 67 68 69 70

eicosanoic acid 17-hydroxyoctadecanoic acid 7-oxo-dehydroabietic acid docosanoic acid tetracosanoic acid hexacosanoic acid shoreic acid oleanonic acid ursonic acid-like molecule ursonic acid oleanonic acid-like molecule ursonic acid-like molecule ursonic acid-like molecule


Figure 10. Total ion chromatogram of sample B neutral fraction. The identified compounds are reported in Table 11. Figure 9. Total ion chromatogram of the acid fraction of the sample B. The identified compounds are reported in Table 10.

The analysis of the acidic fraction reveals the presence of several compounds (the chromatogram is reported in Figure 9, and peak assignment in Table 10). The presence of 15-hydroxyhexadecanoic acid, of 17-hydroxyoctadecanoic acid, and of long chain fatty acids, with tetracosanoic acid being the most abundant, suggest the presence of beeswax. This is confirmed by the analysis of the neutral fraction (Figure 10, Table 11) where the characteristic beeswax long-chain hydrocarbons, alcohols and diols, were highlighted.32 Moreover, the presence of didehydroabietic acid, dehydroabietic acid and 7-oxo dehydroabietic acid leads to the identification of a Pinaceae resin. In the chromatogram, at high retention times (34 min < rt < 39 min), triterpenoid compounds are also present. In particular, shoreic (eichlerianic) acid, oleanonic acid, and ursonic acid, which are known as biomarkers of dammar resin60,61 occur. Its presence is confirmed by the analysis of the neutral fraction where nor-β-amyrone, nor-R-amyrone, 20,24-epoxy-25hydroxydammaren-3-one, hydroxydammarenone, 20,24-epoxy-25hydroxydammaren-3-ol, oleanonic aldehyde, and ursonic aldehyde 4498 Analytical Chemistry, Vol. 78, No. 13, July 1, 2006

were identified. The presence of all these compounds, some of which are known to be subjected to degradation in the course of aging,66-70 suggests that the resin was applied in a relatively recent restoration. Cholesterol and a cholesterol degradation product were also highlighted in the neutral fraction. Their detection may lead to identifying fats of animal origin and confirms the hypothesis on the presence of egg performed by the analysis of the amino acids content. The most abundant peaks in the chromatogram of the acidic fraction are palmitic, stearic, suberic, azelaic, and sebacic acids. Such a high quantity of dicarboxylic acids (ΣD ) 39.5%), together with the characteristic ratios (A/P ) 1.0, P/S ) 1.4) suggest that an aged drying oil is present in the sample.41 Despite the presence (66) Van der Dolen, G. A.; van der Berg, K. J.; Boon, J. J. Stud. Conserv. 1998, 43, 249-264. (67) Van der Doelen, G. A.; Van Der Berg, K. J.; Boon, J. J.; Shibayama, N.; De la Rie, E. R.; Genuit, W. J. L. J. Chromatogr. A 1998, 809, 21-37. (68) Zumbuhl, S.; Knochenmuss, R.; Wulfert, S.; Dubois, F.; Dale, M. J.; Zenobi, R. Anal. Chem. 1998, 70, 707-715. (69) Dietemann, P.; Kalin, M.; Zumbuhl, S.; Knochenmuss, R.; Zenobi, R.; Wulfert, S. Anal. Chem. 2001, 73, 2087-2096. (70) Dietemann, P.; Edelmann, M. J.; Meisterhans, C.; Pfeiffer, C.; Zumbuhl, S.; Knochenmuss, R.; Zenobi, R. Helv. Chim. Acta 2000, 83, 1766-1777.

Table 11. Compounds Identified in Sample B Neutral Fractiona peak no.

compound

71 72 73 74 24 75 26 76 77 78 28 79 80 81 30 82 83 84 85 38 86 39 87 32 88

heneicosane triacosane pentacosane eptacosane tetracosanol enneacosane hexacosanol 1,23-tetracosandiol untriacontane 2,R-methyl-5,R-cholest-3-ene octacosanol cholesterol 1,25-hexacosandiol nor-β-amyrone triacontanol tor-R-amyrone 1,27-octacosandiol nor-β-amyrone-like molecule nor-R-amyrone-like molecule 20,24-epoxy-25-hydroxydammaren-3-one hydroxydammarenone 20,24-epoxy-25-hydroxydammaren-3-ol oleanonic aldehyde dotriacontanol ursonic aldehyde


of beeswax and egg, the P/S and A/P ratios are consistent with the presence of a linseed oil, indicating that egg and beeswax are minor components in the mixture. The various materials identified in this sample would seem to indicate that animal glue was used for the preparation layer, that linseed oil and egg were used as binding media (probably mixed to form a “tempera grassa”), and that the Pinaceae resin belongs to the original varnish (probably mixed with linseed oil). Beeswax and dammar would seem to be restoration materials because the encaustic technique is not consistent with a painting of the 17th18th centuries and dammar resin was introduced into Europe in the 19th century.3 Painted Etrurian Sarcophagus, 4th Century, Sample C. The analysis of the amino acid fraction highlighted the presence of proteinaceous material (see Table 9). Once again, the hydroxyproline marker indicates the presence of animal glue in the sample, and the PCA analysis (Figure 8) does not assign the sample to any cluster. Also in this case, due to the position of the sample between the animal glue and the egg clusters, the hypothesis of the occurrence of egg together to animal glue seems reasonable. In the analysis of the acidic fraction (chromatogram reported in Figure 11 and identified peaks in Table 12) many compounds were highlighted. The occurrence of 15-hydroxyhexadecanoic acid, 17-hydroxy octadecanoic acid, tetracosanoic acid, and hexacosanoic acid (acidic fraction) indicates that small amounts of beeswax are present.32 Epilaccishellolic acid, laccishellolic acid, epishellolic acid, shellolic acid, aleuritic acid and its derivative were identified in the acidic fraction, suggesting the presence of shellac.31 Relatively high amounts in the acidic fraction of palmitic and stearic acids,

Figure 11. Total ion chromatogram of sample C acid fraction. The identified compounds are listed in Table 12. Table 12. Compounds Identified in Sample C Acid Fractiona peak no.

compound

89 45 46 47 48 49 5 90 7 91 82 64 93 94 65 95 96 13 15

eptandioic acid lauric acid suberic acid azelaic acid myristic acid sebacic acid palmitic acid butolic acid stearic acid epilaccishellolic acid (or laccishellolic acid) laccishellolic acid (or epilaccishellolic acid) 15-hydroxyhexadecanoic acid epishellolic acid (or shellolic acid) shellolic acid (or epishellolic acid) 17-hydroxyoctadecanoic acid aleuritic acid derivative aleuritic acid tetracosanoic acid hexacosanoic acid


together with the high content in dicarboxylic acids (suberic, azelaic, and sebacic), suggest the presence of a drying oil. The values of the characteristic ratios (A/P ) 1.3, P/S ) 2.6, ΣD ) 53%) confirm the presence of a drying oil. Moreover in the neutral fraction, cholesterol has been evidenced, confirming the hypothesis of the occurrence of the egg protein performed by the amino acids analysis. Being a drying oil and egg simultaneously present, the source of the oil cannot be identified: the P/S value is in fact consistent both with a mixture of egg and linseed oil or egg and walnut oil, and only the occurrence of poppy seed oil can be excluded. Since the work was painted in the 4th century B.C., the animal glue in the sample was most probably used in the preparation layer, and egg was used as a binder to disperse pigments. The drying oil, beeswax, and shellac must thus have been used for restoration. CONCLUSIONS An analytical procedure for the simultaneous characterization of drying oils, animal and plant terpenoid resins, pitch and tars, natural waxes, and proteinaceous binders on the same microsample by means of GC/MS has been proposed. Proteinaceous binders are identified on the basis of the amino acid profile Analytical Chemistry, Vol. 78, No. 13, July 1, 2006

4499

Table 13. Organic Materials Identified in the Paint Samples from Works of Art sample painting on the hull of a shipwreck, late 1700s/early 1800s: sample A easel painting, 18th-beginning of 19th centuries: sample B painted Etrurian sarcophagus, 4th century: sample C

proteinaceous material

drying oil

natural wax

linseed oil animal glue and egg animal glue and egg

obtained after microwave-assisted acid hydrolysis followed by derivatization with a silylating agent and GC/MS analysis. Moreover, the use of PCA on amino acid relative percentage content may contribute to distinguishing between various proteinaceous materials, namely, egg, casein, and animal glue. Drying oils, waxes, and resins are determined on the residue of the ammonia extraction added to the ether extract of the acidic water solution arising from acidic hydrolysis, after a saponification/ salification step assisted by microwaves, followed by solvent extraction, derivatization with a silylating agent and GC/MS analysis. With the yields of the chemolysis steps being almost quantitative, fractionation of analytes between different phases is avoided, the evaluation of the chromatographic profiles results much easier, and quantitation is reliable. Drying oils and lipids from egg are identified by means of characteristic parameters (A/ P, P/S, ΣD) and waxes and resins on the basis of the evaluation of the chromatographic profiles and on the presence of specific molecular biomarkers. The application of the proposed analytical procedure to reference materials shows that it is suitable for the simultaneous characterization of several classes of compounds and thus, for the identification of proteinaceous material, drying oils, beeswax, plant resins, and shellac in old samples. The quantitative analysis of amino acids and fatty acids, together with the careful evaluation of blanks, ensure the data quality and permits to reliably characterize proteins and lipids, distinguishing from environmental contaminations. The ability to recognize several materials is due to the use of molecular markers that remain stable under aging or are stable products of aging. The fact that these compounds can be detected at the nanogram concentration level means that heterogeneous


plant resin

animal resin

pitch from Pinaceae plants

linseed oil

beeswax

yes

beeswax

Pinaceae resin and dammar resin shellac

samples, whose organic contents are only few tenths of micrograms, can be characterized. The suggested procedure allows to collect small sized samples from painted works of art for a successful chemical characterization: thus, it may be considered a micro-invasive technique for organic material identification. The procedure has been used to characterize three samples coming from a painting on a shipwreck of the 17th -18th century (wood support), an easel painting of the 17th-18th century (canvas support), and an Etruscan painted sarcophagus, 4th century B.C. (calcareous alabaster support). Animal glue, egg, linseed oil, beeswax, Pinaceae resin, dammar, and shellac were the identified materials found in mixtures, and the results are summarized in Table 13. These data permit us to identify some restoration materials (as shellac and dammar), allow us to achieve a deeper knowledge on the painting techniques, and are a fundamental tool for the choice of a proper conservative intervention. ACKNOWLEDGMENT We thank Dr. Angelo Bottini of the Soprintendenza ai Beni Archeologici per la Toscana (Florence) for involving our group in the study of the “Sarcofago delle Amazzoni”. We also thank Dr. Yaacov Kahanov of The Leon Recanati Institute for Maritime Studies and the University of Haifa (Haifa, Israel) for having provided the samples from the wreck, and for stimulating discussion on the results. PRIN2003 (MURST) is acknowledged for the research funds. Received for review November 3, 2005. Accepted April 7, 2006. AC0519615

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