MS Analytical Procedure for the Characterization of Glycerolipids

Anal. Chem. 2010, 82, 376–386

GC/MS Analytical Procedure for the Characterization of Glycerolipids, Natural Waxes, Terpenoid Resins, Proteinaceous and Polysaccharide Materials in the Same Paint Microsample Avoiding Interferences from Inorganic Media Anna Lluveras, Ilaria Bonaduce,* Alessia Andreotti, and Maria Perla Colombini Laboratory of Chemical Science for Safeguarding the Cultural Heritage, Department of Chemistry and Industrial Chemistry, University of Pisa, Via Risorgimento 35, 56126 Pisa, Italy An innovative GC/MS procedure for the characterization of organic materials in samples from works of art was developed. It is based on a multistep chemical pretreatment of the samples based on the ammonia extraction of proteins and polysaccharide materials, in order to separate them from lipid and resinous materials. The extraction is then followed by the separation and purification of proteinaceous and polysaccharide materials before hydrolysis, based on the use of monolithic sorbent tip technology with a C4 stationary phase. Lipids and resins are saponified/salified separately. Three fractions are generated and analyzed separately by GC/MS, thus enabling a quantitative analysis to be performed on aldoses and uronic acids, amino acids, mono- and dicarboxylic aliphatic acids, to determine polysaccharide, proteinaceous, and glycerolipid materials and molecular pattern recognition for the natural resin and wax components. With this analytical procedure, for the first time, glycerolipids, natural waxes, and proteinaceous, resinous, and polysaccharide materials can be simultaneously characterized in the same microsample from painted works of art. This new analytical approach prevents any analytical difficulties arising when the sample is divided into several different aliquots to be chemically processed separately, in order to characterize the various classes of organic materials. The procedure was successfully applied to samples from paintings from the Bamiyan Buddhas and a panel painting from the 15th century, highlighting the occurrence of glycerolipids, animal and plant resins, proteinaceous and polysaccharide materials. From a physicochemical point of view, paintings are composite arrays of several layers made of a heterogeneous mixture of organic (binding media, varnishes, colorants) and inorganic materials (pigment, thickeners, stabilizers, dryers, and extend* To whom correspondence should be addressed. Tel: +390502219252. Fax: +390502219260. E-mail: [email protected].

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ers).1 These materials undergo interaction with each other, as well as exposure to pollutants, light, and thermo-hygrometric conditions. Natural organic materials used as binding media (milk, casein, animal glue, linseed oil, blood, beeswax, plant gums, honey, plant resins, etc.) are complex mixtures consisting of proteins, triglycerides, terpenoid compounds, sterols, hydrocarbons, aliphatic esters, aliphatic alcohols, fatty acids, polysaccharides, etc. With aging these compounds may be subject to oxidation, hydrolysis, and cross-linking reactions, which modify the original composition of the material.2,3 As a result, paintings are complex evolving systems, the characterization of which represents a difficult task for the analytical chemist. GC/MS analytical techniques are best suited for identifying natural organic materials used as paint binders,2 even though proteomics proved to be extremely promising in the characterization of proteinaceous media.4 Several GC/MS analytical procedures for characterizing organic materials present in painting samples as binders and varnishes have been developed in recent years.,2,3,5 and references therein. Most compounds present in natural organic binders are macromolecular and polar. Their analysis by GC/MS thus entails chemolysis and derivatization. Purification from inorganic media is also important when dealing with the analysis of proteinaceous and saccharide materials.2 Due to the uniqueness and small dimensions of samples collected from painted works of art, it is fundamental to use an analytical procedure that allows the characterization of as many materials as possible on the same sample. A GC/MS analytical procedure for the characterization of lipids, waxes, and resinous and (1) Mills, J. S.; White, R. The Organic Chemistry of Museum Objects, 2nd ed.; Butterworth Heinemann Ltd: Oxford, 1994. (2) Andreotti, A.; Bonaduce, I.; Colombini, M. P.; Modugno, F.; Ribechini, E. In New Trends in Analytical, Environmental and Cultural Heritage Chemistry; Tassi, L., Colombini, M. P., Eds.; Transworld Research Network: Kerala, 2008; pp 389-423. (3) Scalarone, D.; Chiantore, O. In Organic Mass Spectrometry in Art and Archaeology; Colombini, M. P.; Modugno; F., Eds.; John Wiley & Sons: New York, 2009; pp 327-361. (4) Kuckova, S.; Hynek R.; Kodicek, M. In Organic Mass Spectrometry in Art and Archaeology; Colombini, M. P., Modugno; F., Eds.; John Wiley & Sons: New York, 2009; pp 165-187. (5) Colombini, M. P.; Modugno, F. J. Sep. Sci. 2004, 27, 147–160. 10.1021/ac902141m  2010 American Chemical Society Published on Web 12/02/2009

proteinaceous materials in the same paint microsample is described in the literature.6 The procedure involves a multistep chemical pretreatment of the sample, which leads to the separation of an aqueous fraction containing amino acids and of two organic fractions (acidic and neutral), which are both submitted to silylation prior to the GC/MS analysis. This procedure was recently improved, thus making it reliable when high amounts of inorganic interfering media are present.7 This was achieved by using miniaturized C4 solid phases placed on pipet tips in order to purify proteins and peptides. However, until now, polysaccharide materials have always had to be determined on a different sample aliquot.8 Only one analytical procedure is available for the simultaneous characterization of polysaccharide and proteinaceous materials in the same sample,9 but it does not enable any information on its lipid and resinous content to be obtained. The analysis of sugars by GC/MS generally encounters several analytical difficulties,10-12 the main one being an efficient derivatization. In water solutions, monosaccharides undergo intramolecular reactions to form cyclic hemiacetals and hemiketals, both five- and six-membered rings, which are in equilibrium with each other. To avoid the formation of multiple chromatographic peaks,13 causing irreproducible quantification, loss of sensitivity, and unreliable identifications, various strategies have been described: (1) reducing carbonyl moieties to hydroxymethyl groups followed by acetylation14 results in the formation of only one derivative from more than one sugar (for instance, mannitol is formed from the reduction of both mannose and fructose); (2) converting the parental monosaccharides into an acyclic oxime followed by silylation or acetylation12,15,16 leads to the formation of two derivatives (syn and anti); and (3) mercaptalation followed by silylation of the parental sugars.8,17 Although this procedure seems extremely promising, since only one peak for each analyte is obtained, laboratory practice has led to several problems: variable yields are obtained, and in some cases, the formation of byproduct makes the derivatization reaction inefficient, thus leading to the contamination of the chromatographic system. In this paper, the optimization of the procedure outlined in refs 8 and 17 is presented. It involves acidic hydrolysis assisted by microwaves to free the sugars, a cleanup step of the sample to eliminate the inorganic material, and a derivatization consisting of a mercaptalation followed by silylation. Important modifications were introduced in the cleanup and derivatization steps to avoid the above-mentioned problems. (6) Andreotti, A.; Bonaduce, I.; Colombini, M. P.; Gautier, G.; Modugno, F.; Ribechini, E. Anal. Chem. 2006, 78, 4490–4500. (7) Bonaduce, I.; Cito, M.; Colombini, M. P. J. Chromatogr. A 2009, 1216, 5931–5939. (8) Bonaduce, I.; Brecoulaki, H.; Colombini, M. P.; Lluveras, A.; Restivo, V.; Ribechini, E. J. Chromatogr. A 2007, 1175, 275–282. (9) Schneider, U.; Kenndler, E. Fresen. J. Anal. Chem 2001, 371, 81–87. (10) Wiliams, P. A.; Philips, G. O.; Stephen, A. M.; Churms, S. C. Gums and Mucilage, Food Polysaccharides and Their Applications, 2nd ed.; CRC Press Taylor and Francis Group: Phiadelphia, 2006; pp 455-495. (11) Sanz, M. L.; Nartı´nez-Castro, I. J. Chromatogr. A 2007, 1153, 74–89. (12) Molna´r-Perl, I. J. Chromatogr. A 2000, 891, 1–32. (13) Mejanelle, P.; Bleton, J.; Tchapla, A.; Goursaud, S. J. Chromatogr. Libr. 2002, 66, 845–902. (14) Blakeney, A. B.; Harris, P. J.; Henry, R. J.; Stone, B. A. Carbohyd. Res. 1983, 113, 291–299. (15) Pitthard, V.; Griesser, M.; Stanek, S.; Bayerova, T. Macromol. Symp. 2006, 238, 37–45. (16) Pitthard, V.; Griesser, M.; Stanek, S. Ann. Chim. 2006, 96, 561–573. (17) Pitthard, V.; Finch, P. Chromatogr. Suppl. 2001, 53, 317–321.

This analytical procedure was then used to set up an innovative GC/MS analytical procedure for the determination of glycerolipids, natural waxes, terpenoid resins, proteinaceous and polysaccharide materials in the same paint microsample in the presence of interfering inorganic materials. To our knowledge, this is the only GC/MS analytical procedure reported in the literature capable of characterizing, in the same microsample, the main classes of natural organic materials that can be encountered as binders or varnishes in a paint sample. With this analytical procedure, the separation between lipid, resinous, polysaccharide, and proteinaceous materials is achieved, and the purification of proteinaceous binders is performed simultaneously. This analytical procedure enables (1) proteinaceous binders (egg, collagen, casein, garlic) to be identified on the basis of the quantitative determination of the amino acid profile processed by principal component analysis; (2) glycerolipids (linseed oil, poppy seed oil, walnut oil, and egg) to be identified on the basis of the quantitative determination of fatty and dicarboxylic acids; (3) plant resins (Pinaceae resins, sandarac, mastic, and dammar), animal resins (shellac), tar, or pitches and natural waxes (beeswax, carnauba wax) to be identified on the basis of the molecular pattern recognition of aliphatic long chain alcohols, midlong-chain acids, hydrocarbons, and terpenic molecular markers; and (4) polysaccharide binders (tragacanth, arabic, and fruit tree gums) to be identified on the basis of the quantitative/qualitative determination of occurring sugars. The successful application of this procedure for the analysis of a sample from the Eastern Buddha (Bamiyan, Afghanistan) from the 6th century and a panel painting from the 15th century will be also shown and the identification of organic components discussed. 1. EXPERIMENTAL SECTION 1.1. Reagents. All the solvents were Baker HPLC grade and were used without any further purification. Trifluoroacetic acid (99% purity) and anhydrous pyridine were from Fluka (Milan, Italy). Ethanethiol (ETSH; 99.5%), sodium azide (NaN3; 99.5%), N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with and without 1% trimethylchlorosilane, N-tert-butyldimethylsilyl-Nmethyltrifluoroacetamide (MTBSTFA) with 1% trimethylchlorosilane, and triethylamine were purchased from Sigma-Aldrich. The following solutions, apart from those for the amino acids, were prepared by weighing pure substances and were used as standards: (i) amino acid solution in 0.1 M HCl, purchased from Sigma-Aldrich 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) solution of fatty and dicarboxylic acids 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 >99%) were purchased from Sigma-Aldrich]; (iii) norleucine solution in bidistilled water (Sigma-Aldrich; purity 99%, 138.66 µg/g) was used as a derivatization internal standard for amino acids; (iv) tridecanoic acid Analytical Chemistry, Vol. 82, No. 1, January 1, 2010

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solution in isooctane (Sigma-Aldrich; purity 99%, 135.48 µg/g) was used as a lipid-resinous fraction derivatization internal standard; (v) hexadecane solution in isooctane (Sigma-Aldrich; purity 99%, 80.34 µg/g) was used as an injection internal standard; (vi) monosaccharides and uronic acids solution in bidistilled water containing D-(+)-galactose (0.1 mg/g), L-(-)-fucose (0.1 mg/g), L-(+)-arabinose (0.1 mg/g), L-(-)-ramnose (0.1 mg/g), L-(-)mannose (0.1 mg/g), D-(+)-xylose (0.1 mg/g), D-(+)-glucose (0.1 mg/g), D-glucuronic acid (0.1 mg/g), D-galacturonic acid (0.1 mg/ g) monohydrate; and (xi) mannitol in bidistilled water (0.1 mg/g) was used as a derivatisation internal standard for aldoses and uronic acids. All monosaccharides and uronic acids (purity 99%) were purchased from Sigma-Aldrich (Milan, Italy). All standard solutions were stored at 4 °C. 1.2. Apparatus. A microwave oven model (MLS-1200 MEGA Milestone, FKV, Sorisole, Bergamo, Italy) was used for the hydrolysis of proteins and peptides, and the saponification/ salification of glycerolipid, waxy, and resinous materials. Operating conditions are detailed in section 1.4. An RC10-22 speed vacuum system with a refrigerated ion trap RCT 90 from Thermo Electron Corp. (St. Herblain, France) was used to dry the water solutions. A Fourier transform infrared spectrometer (Thermo Scientific Nicolet 380, Waltham) was equipped with a DTGS detector. The spectrometer has a geranium on KBr beamsplitter and a EverGlo source. The spectra were the sum of four scans collected from 4500 to 400 cm-1 at a resolution of 2 cm-1. The OMIX C4 pipet tips were purchased from Varian (Milan, Italy). They consist of 100 µL capacity pipet tips, containing a miniaturized solid-phase extraction bed of functionalized monolithic sorbents, inserted inside the tips. The cation/anion exchange resin Zerolit DMF, with the inclusion of an indicator and granulometry, comprised between 14 and 52 mesh, was supplied by BDH Chemicals Ltd. (UK). A 6890N GC System gas chromatograph (Agilent Technologies) equipped with PTV injector was coupled with a 5975 mass selective detector (Agilent Technologies) single golden quadrupole mass spectrometer. 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 at 150 °C. Chromatograms were acquired both total ion chromatogram (TIC) mode and selected ion monitoring (SIM) mode. For the gas chromatographic separation an HP-5MS fused silica capillary column (5% diphenyl-95% dimethylpolysiloxane, 30 m × 0.25 mm i.d., 0.25 µm film thickness, J&W Scientific, Agilent Technologies, Palo Alto, CA) coupled with a deactivated silica precolumn (2 m × 0.32 mm i.d., J&W Scientific, Agilent Technologies, Palo Alto, CA) using a quartz press fit was used. The carrier gas was used in the constant flow mode (He, purity 99.995%) at 1.2 mL/min for the amino acid and the lipid-resinous fractions and at 1.0 mL/min for the saccharide fraction. The GC/MS parameters for the analysis of the different fractions are as follows: Lipid-Resinous Fraction. The PTV injector was used in splitless mode at 300 °C and 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 378


isothermal for 3 min, 20 °C/min up to 300 °C isothermal for 30 min. Amino Acids Fraction. The PTV injector was used in splitless mode at 220 °C, and the chromatographic oven was programmed as follows: initial temperature 100 °C isothermal for 2 min, then 4 °C/min up to 280 °C, 280 °C isothermal for 15 min. Saccharide Fraction. The PTV injector was used in splitless mode at 250 °C and the chromatographic oven was programmed as follows: 50 °C isothermal for 2 min, 5 °C/min up to 190 °C at, 190 °C isothermal for 20 min 5 °C/min up to 280 °C, 280 °C isothermal for 15 min. 1.3. Samples. 1.3.1. Raw Materials. Arabic and tragacanth gums were purchased from Sigma-Aldrich. Cherry gum was kindly provided by the Opificio delle Pietre Dure (Florence, Italy). Materials were analyzed from the uniformly grinded solid. 1.3.2. Reference Painting Layers. Reference paint layers of polysaccharide gums (arabic, tragacanth, and fruit tree gums) were prepared dissolving the gums in bidistilled water and applying them onto a glass support. Sodium azide (NaN3) was admixed as biocide. 1.3.3. Paint Samples. The paint samples came from the Eastern Buddha (Bamiyan, Afghanistan), 6th century, and from a panel painting from the 15th century. The panel painting was covered by a dark patina, which was an oxidized varnish applied on top of the painting in an undocumented past restoration. The choice of the appropriate technique to remove the patina is related to its chemical composition. The sample weight was 0.1 mg. The Buddhas of Bamyan were two monumental statues of standing Buddhas carved into the side of a cliff in the Bamyan valley in the Hazarajat region of central Afghanistan. The main bodies were carved directly from the stone, but details were modeled in clay. The resulting sculptures were painted. Since their destruction in 2001, paint fragments have been recovered and have been subjected to analytical investigations, under the direction of UNESCO. In this paper, an investigation performed on the organic binders of one of these samples (sample 188) is presented. The fragment showed four layers: the clay, the plaster layer applied as a preparation, and two painted layers, one white and one yellow. The painted layers were sampled by scraping the fragment with a scalpel under a binocular microscope. Due to their high inorganic content, the subsamples collecteds188-3 representing the white layer and 188-4 representing the yellow layerswere relatively big, 1.7 and 1.2 mg, respectively. 1.4. Analytical Procedure. The overall combined analytical procedure for the determination of glycerolipids, terpenoid resins, natural waxes, proteinaceous and polysaccharide materials, also in the presence of high amounts of interfering inorganic pigments dryers and extenders, is presented in Figure 1. The procedure is as follows. (I) The sample is subjected to ammonia extraction. In order to solubilize proteins and saccharides and to separate them from insoluble inorganic salts, 200-400 µL of 2.5 M NH3 is added to the sample in an ultrasonic bath at 60 °C for 120 min, twice. During this step, free organic acids soluble in ammonia are extracted together with the proteinaceous matter and polysac-

Figure 1. The GC/MS combined analytical procedure.

charide materials. The residue containing insoluble organic and inorganic species is kept for step XII. (II) The extracted ammonia solution (proteinaceous-polysaccharide fraction) is evaporated to dryness under a stream of nitrogen and redissolved in 100 µL of 1% TFA. (III) The acidic solution, containing proteins, peptides, saccharides, soluble salts, and free organic acids, is extracted with diethyl ether (200 µL, three times). The free organic acids extracted together with proteins and polysaccharides 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 XII. (IV) The acidic solution containing proteins, peptides, polysaccharides, and soluble inorganic salts is fluxed with nitrogen to remove the excess of ether and then applied onto a Omix C4 tip using 10 aspirating/dispensing cycles; 0.1% TFA is used as rinsing solution (100 µL, twice) and formic acid (0.1%)/MeOH (75%)/ H2O (25%) as eluting solution (100 µL, twice). The rinsing solution admixed with the residue of the purification step are kept for step V. The eluted solution is kept for step IX. (V) The residue admixed with the rinsing solution of the OMIX C4 purification is a solution of soluble inorganic salts, along with any proteinaceous materials that exceed the capacity of the tip, and polysaccharide materials. It is transferred into closed PTFE conic vials, dried under nitrogen stream, admixed with 0.5 mL of 2 M trifluoroacetic acid and then undergoes microwave-assisted acid hydrolysis. Hydrolysis conditions are as follows: power 500 W, temperature 120 °C, duration 20 min. (VI) After hydrolysis, the solution is filtered with a PTFE membrane and then dried in a rotatory evaporator. Once reconstituted in a 100 µL of bidistilled water, the solution of the freed sugars is purified on a Zeolit DMF double-exchange resin, packed on a 0.5 cm diameter glass column. Sugars are eluted with 2 mL of bidistilled water. The aqueous phase is a

solution containing purified aldoses and uronic acids: the saccharide fraction. (VII) An aliquot of the solution containing aldoses and uronic acids is added with 5 µL of the mannitol solution, evaporated to dryness in the rotary evaporator, and subjected to a three-step derivatization: one mercaptalation step and two silylation steps. The mercaptalation consists of adding 25 µL of ethanethiol/ trifluoroacetic acid (2/1, v/v) to the sugars and keeping the mixture at room temperature for 10 min with sporadic shaking. Aldoses and uronic acids are transformed into the corresponding diethyl dithioacetals and diethyl dithioacetal lactones. In the first silylation step, 100 µL of BSTFA is added to the mercaptalation mixture and the mixture is kept for 15 min at 60 °C. After drying under a nitrogen flow (to remove the TMS derivatives of TFA and ETSH), the second silylation step consists of adding 50 µL of BSTFA with 1% TMCS as a derivatizing agent and 100 µL of pyridine as a solvent. The reaction mixture is kept at 60 °C for 45 min to completely silylate the mercaptal derivatives of the parental sugars. (VIII) The reaction mixture is then dried under a nitrogen flow and redissolved in 50 µL of hexane; 2 µL of this solution, containing diethyl dithioacetal trimethylsilyl derivatives of the parental sugars, is analyzed by GC/MS. (IX) The solution of highly purified proteins and peptides in formic acid (0.1%)/MeOH (75%)/H2O (25%) is dried under a stream of nitrogen and subjected to acidic hydrolysis assisted by microwaves (power ) 250 W for 40 min) in the vapor phase with 30 mL of 6 M HCl at 160 °C for 40 min. After the hydrolysis, bidistilled water (200-400 µL) is added to the acidic hydrolysate. The aqueous phase is an acidic solution containing highly purified amino acids: the amino acid fraction. (X) An aliquot of the amino acid fraction, admixed with 5 µL of norleucine solution and 10 µL of hexadecane solution, is evaporated to dryness under a stream of nitrogen and undergoes Analytical Chemistry, Vol. 82, No. 1, January 1, 2010

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derivatization with 20 µL of MTBSTFA, 40 µL of pyridine (solvent), and 2 µL of triethylamine (catalyst), at 60 °C for 30 min. (XI) A total of 2 µL of the pyridine solution of derivatized amino acids is analyzed by GC/MS. (XII) The residue of the ammonia extraction, combined with the ethereal extracts, undergoes microwave-assisted saponification/salification with 300 µL of 10 wt % KOH in ETOH at 60 °C for 120 min. (XIII) After saponification, the alcoholic solution is diluted in bidistilled water and acidified with trifluoroacetic acid (aqueous solution 1:1). (XIV) The acidic solution is then extracted with hexane (200 µL, three times) and, afterward, with diethyl ether (200 µL, three times). The extracts are dried under a nitrogen flow (to remove trifluoroacetic acid, which is partially soluble in the organic phase) and redissolved in acetone and hexane (300 µL 1:1): the lipid-resinous fraction. (XV) An aliquot of the lipid-resinous fraction is admixed with 5 µL of tridecanoic acid solution, evaporated to dryness under a nitrogen flow, and derivatized with 20 µL of BSTFA and 200 µL of isooctane (solvent) at 60 °C for 30 min. Lastly, 5 µL of the hexadecane solution is added. (XVI) A total of 2 µL of the isooctane solution of acidic and neutral compounds, derivatized if containing OH moieties, is analyzed by GC/MS. The analysis of this fraction enables acidic and neutral terpenoid compounds, sterols, alcohols, alkanes, monocarboxylic acids, dicarboxylic acids, and hydroxy acids to be determined. The quantitative determination of amino acids, aldoses and uronic acids, aliphatic mono- and dicarboxylic acids is performed by using standard solutions, building calibration curves, and evaluating daily recoveries. Running blanks of the procedure highlighted a low level of contamination. The detection limit (LOD) and the quantitation limit (LOQ) of amino acids, aldoses, uronic acids, and fatty and dicarboxylic acids were calculated. At a statistical significance level of 0.05, the LODs and LOQs obtained are reported in Table 1. 2. RESULTS AND DISCUSSION 2.1. Combined Analytical Procedure. Analyzing the polysaccharide, proteinaceous, and lipid resinous materials in the same sample entails separating three different fractions, to be chemically processed in different ways: (1) proteinaceous materials require hydrochloric acid 6 M as a hydrolyzing agent, and microwaveassisted hydrolysis is performed in open glass conic vials (power 250 W, temperature 160 °C, duration 40 min); (2) polysaccharide materials require trifluoroacetic acid 2 M as a hydrolyzing agent, and microwave-assisted hydrolysis is performed in closed PTFE conic vials (power 500 W, temperature 120 °C, duration 20 min); (3) lipid resinous materials require salification/saponification with 10% KOH in ethanol, and the microwave-assisted reaction is performed in closed PTFE conic vials (power 200 W, temperature 80 °C, duration 60 min). In addition, proteinaceous materials require a cleanup step, to remove any inorganic materials that could interfere with the GC/ MS analyses. Thus, to determine the polysaccharide materials in the same sample used for the determination of proteinaceous and lipid resinous materials, it is necessary (1) to optimize the GC/ 380


Table 1. LOD and LOQ of Fatty Acids, Dicaboxylic Acids, Amino Acids, Aldoses, and Uronic Acids compound

LOD/ng

LOQ/ng

alanine (Ala) glycine (Gly) valine (Val) leucine (Leu) isoleucine (Ile) serine (Ser) proline (Pro) phenylalanine (Phe) asparagine (Asp) glutamic Acid (Glu) hydroxyproline (Hyp) lauric acid suberic acid myristic acid sebacic acid azelaic acid palmitic acid oleic acid stearic acid xylose (Xyl) arabinose (Ara) ramnose (Ramn) fucose (Fuc) galacturonic acid (GalAc) glucuronic acid (GlucAc) glucose (Glu) mannose (Mann) galactose (Galact)

8 16 7 14 7 12 9 5 10 18 0 12 15 24 71 3 902 7 1712 130 20 10 10 0 0 440 110 30

12 24 11 24 12 24 14 9 19 36 0 20 29 46 91 7 1478 13 2833 270 50 20 20 0 0 850 200 60

MS analytical procedure for the characterization of polysaccharide binders (section 2.1.1); (2) to evaluate the behavior of plant gums when subjected to the ammonia extraction used to separate proteins from lipids and resins (section 2.1.2); and (3) if polysaccharide materials are extracted in ammonia together with proteins, it is then necessary to evaluate the behavior of plant gums when subjected to the purification procedure used to eliminate inorganic materials from the proteins using the C4 stationary phase (section 2.1.3). 2.1.1. Optimization of the GC/MS Analytical Procedure for the Characterization of Polysaccharide Binders. The analytical procedure in ref 8 was improved by optimizing the cleanup and derivatization steps. 2.1.1.1. Cleanup Step. When analyzing polysaccharide materials in paint samples by GC/MS, cleanup is necessary prior to the chromatographic analysis in order to suppress any interference due to inorganic components.8 According to the analytical procedure previously published, the solid sample is subjected to microwave-assisted hydrolysis. The hydrolysated solution is then filtered to remove insoluble material, cleaned up using a strong cation exchange resin to suppress any interference from cations, and subsequently derivatized.8 In reality, there were some derivatization problems in the analyses of samples with a high content of sulfates and silicates (data not shown), as revealed by the very low intensity peaks of all the sugars, including mannitol (the derivatization internal standard), observed in the chromatograms. This would seem to highlight that anions can also interfere. Replacing the strong cation exchange resin with a double exchange resin, both cations and anions are simultaneously removed from the solution containing the sugars that were freed from the hydrolysis step.

The procedure is thus modified as follows: the solid sample undergoes microwave-assisted hydrolysis. It is then filtered to remove any insoluble material, dried under a nitrogen flow in order to eliminate trifluoroacetic acid, reconstituted in water, and then applied to a double exchange resin. The purified solution of aldoses and uronic acids freed from the plant gums can thus be derivatized before undergoing GC/MS analysis. 2.1.1.2. Derivatisation Procedure. The derivatization procedure8 can be summarized as follows: 25 µL of a 2:1 ETSH:TFA mixture is added to the dried solution of free aldoses and uronic acids, leading to the formation of the corresponding diethyl dithioacetals and diethyl dithioacetal lactones. After 20 min, 80 µL of BSTFA and 50 µL of pyridine are added at 60 °C to persilylate the diethyl dithioacetals and diethyl dithioacetal lactones. After 45 min the solution is dried under a nitrogen flow, reconstituted in hexane, and injected into the GC/MS. Although the procedure avoids the formation of more than one peak per analyte when aldoses and uronic acids are analyzed, drying the mixture prior to the GC injection was problematic. In fact, the drying itself tended to take a long time (30-45 min), and in some cases precipitates formed, or the solution would not dry at all. In these latter cases, the derivatization was occasionally ineffective, leading to an irreversible contamination of the chromatographic system. One possible explanation could be the formation of pyridine trifluoroacetate (PyTFA) in the reaction mixture. To find out if the formation of PyTFA took place, the FTIR spectrum of a mixture of 8 µL of TFA with 50 µL of pyridine (the ratio corresponding to the derivatization mixture) was recorded (Figure 2A). It presented all the characteristic bands of pyridine (600, 705, 740, 990, 1025, 1070, 1145, 1215, 1435, 1480, 1580, 1600, 1875, 1923, 1995, 3010, 3035, 3060, 3085, and 3150 cm-1) plus some bands that did not correspond to the pyridine or to the TFA spectra (580, 675, 780, 1120, 1200, 1250 sh, 1840, 3650 cm-1). These bands are in agreement with the reference spectra for pyridine trifluoroacetate (1680, 1645, 1560, 1490, 1460, 1420, 1384, 1260, 1195, 1180, 1070, 1040, 990, 950, 840, 802, 760, 722, 683, 604 cm-1). In addition, drying this mixture under a nitrogen flow (30 min) produced a white solid. The FTIR spectrum of this solid presented all the characteristic peaks of pyridine trifluoroacetate, thus confirming that this compound had not been completely removed when drying the reaction mixture (Figure 2B). Lastly, also in the case of the complete reagent mixture (i.e., 25 µL of EtSH:TFA (2:1), 50 µL of pyridine, 80 µL of BSTFA) the peaks characteristic of pyridine trifluoroacetate were identified in the FTIR spectrum (Figure 2C). The occurrence of pyridine trifluoroacetate in the final reaction mixture may explain the difficulties observed in the drying processes and the chromatographic problems arising when the solution was injected into the GC. In fact, the temperature, the drying speed, and the sample composition may all influence the equilibrium (pyridine + TFA T pyridine trifluoroacetate). As a result, when drying, the removal of pyridine and TFA from the reaction mixture might not be enough to guarantee the displacement of the equilibrium to the left and, thus, the formation of TFA and pyridine from the pyridine trifluoroacetate. The occurrence of pyridine trifluoroacetate must therefore be considered

Figure 2. Spectra obtained for (A) a mixture of pyridine and TFA (6:1); (B) solid obtained after drying the mixture pyridine-TFA (6:1); (C) mixture of pyridine, ETSH, TFA, and BSTFA (2:1:6:10). (*) The characteristic bands of pyridine trifluoroacetate, which are listed in the text.

as responsible for the incomplete reaction yields in the subsequent silylation step observed. Pyridine is fundamental in ensuring the completeness of the silylation reaction, as reported in refs 18 and 19 and as highlighted by an average relative standard deviation of the sugar peak areas of 9% that was obtained when pyridine was used, versus 20% when pyridine was not used.8 Since pyridine has to be used, TFA and ETSH must be removed from the reaction mixture prior to the addition of pyridine, to avoid the coexistence of TFA and pyridine in the reaction mixture. To do so, a three-step derivatization was tested. Since the highest mercaptalation yields are obtained after short reaction

(18) Hsu, C.; Cheng, C.; Lee, C.; Ding, W. Talanta 2007, 72, 199–205. (19) Evershed., R. P. In Handbook of Derivatives for Chromatography, 2nd ed.; Blau, K., Halket, J., Eds.; John Wiley & Sons: Chichester, England, 1993.


381

Figure 3. Fucose (deoxyhexose) derivatized by (A) silylation only and (B) mercaptalation followed by silylation. Glucuronic acid derivatized by (C) silylation only and (D) mercaptalation and silylation. From the silylation four peaks can be observed corresponding to the four forms of the sugar in equilibrium in the solution. Silylation conditions in both cases were as follows: 45 min, 60 °C, 100 µL of BSTFA, 50 µL of pyridine. Persilylated sugars are indicated with *. Derivatization byproducts are indicated with (. I.S. corresponds to the internal standard (mannitol).

times (10-20 min) at room temperature20 and byproduct are obtained for long reaction times, by adding BSTFA to the mercaptalation mixture after 10 min, the mercaptalation reaction is stopped suddenly, and TFA and ETSH are derivatized (15 min at 60 °C). In this way, TFA trimethylsilyl ester and ETSH trimethylsilyl ether can be quickly evaporated by drying the reaction mixture under a nitrogen flow. To ensure the completeness of the silylation of the mercaptal derivatives of the parental sugars, another silylation step using pyridine as a solvent is still necessary. Using a mixture of BSTFA:Py > 1 shows that only aldo pentoses (xylose and arabinose) generate just one peak, while chromatograms of methyl hexoses, hexoses, and uronic acids presented from two to five peaks, which were the main peaks corresponding to the trimethylsylilated diethyl dithioacetal of the parental sugar and the others unidentified. Figure 3 shows the chromatogram obtained from a deoxy hexose (fucose) and an uronic acid (glucuronic acid), clearly indicating that the peaks (20) Horton, D.; Norris, P. Preparative Carbohydrates Chemistry, Hanessian, S., Ed.; CRC Press: Boca Raton, FL, 1997; pp 35-53 (ISBN 0824798023).

382


Figure 4. Chromatograms obtained by modifying the conditions in the second silylation step (45 min at 60 °C): (A) fucose, BSTFA: pyridine (2:1); (B) fucose, BSTFA (1% TMCS):pyridine (1:2) (C) glucuronic acid, 100 µL of BSTFA, and 50 µL pyridine (2:1); (D) glucuronic acid, BSTFA (1% TMCS):pyridine (1:2). Persilylated sugars are indicated with *. Derivatization byproduct are indicated with (. I.S. corresponds to the internal standard (mannitol).

corresponding to unidentified compounds do not correspond to the persilylated derivatives of the parental sugar. This meant that the silylation of mercaptalated sugars was the step needing improvement. To this aim, catalytic amounts of TMCS were added and various BSTFA:pyridine ratios were investigated. Using a ratio of 2:1, 5:1, 3:1, and 1:1 did not introduce any significant improvement. Using 50 µL of BSTFA with 100 µL of pyridine (1:2) and 1% TMCS as a catalyst, just one peak was obtained in the chromatogram of all pentoses, hexoses, and methyl hexoses. Chromatograms of the uronic acids still presented some smaller peaks of the above-mentioned unidentified compounds, but the uronic acid derivatization byproduct ratio was g10. Figure 4 shows the chromatograms obtained after the modification of the second silylation step for fucose and glucuronic acids. The reproducibility of the derivatization procedure was tested by analyzing five replicates of a sugar standard solution. An RSD < 10% was observed for each sugar. To evaluate the stability of the derivatized solutions, a derivatized sugar standard solution was injected repeatedly and stored at -18 °C. An RSD lower than 14% was obtained for the first 270 min. Figure 5 reports the chromatogram of a standard solution of the seven monosaccharides and the two uronic acids (100 ppm concentration), showing only one peak for each sugar.

Figure 5. Chromatogram of a standard solution of TMS mercaptal derivatives of the nine sugars. I.S. corresponds to the internal standard (mannitol).

Figure 6. Fructose subjected to the three-step derivatization. I.S. corresponds to the internal standard (mannitol). F1-13 are peaks ascribable to fructose subjected to the three-step derivatization procedure.

The procedure described was set up for the analysis of plant gums, i.e., saccharide materials containing aldoses and uronic acids. When ketoses are present in the sample, more than one peak is obtained by this procedure, none of which correspond to the persilylated sugars. In fact, dialkyl dithioacetals of ketoses are not obtained directly from the parent sugar but ketoses undergo decomposition reactions during mercaptalation.20 As an example, Figure 6 presents the chromatogram of fructose, which is a ketohexose, subjected to the three-step derivatization procedure. 2.1.1.3. Analytical Procedure for the Characterization of Polysaccharide BinderssSummary. On the basis of the modifications introduced, Figure 7 outlines the analytical procedure for the characterization of polysaccharide materials in paint samples. 2.1.2. Ammonia Extraction of Polysaccharides. In order to integrate the optimized analytical procedure for the characterization of polysaccharide materials with the procedure for the characterization of lipid, resinous, and proteinaceous materials,7 the ammonia extraction used to separate proteins from lipids and resins had to be tested on polysaccharides. An analysis of reference painting samples of plant gums was performed with and without ammonia extraction in order to evaluate the percentage of gum recovered. The ammonia extractions of arabic gum, fruit tree gum, and tragacanth gum were performed as follows: 200-400 µL of 2.5 M NH3 was added to the sample in an

ultrasonic bath at 60 °C for 120 min, twice. After the extraction, the extracted ammonia solutions were put together, dried under nitrogen flow, hydrolyzed, and derivatized according to the procedure described in Figure 7. The percentage recovery was calculated as the sum of the amounts of aldoses and uronic acids detected, with respect to the weighted amount. Data are presented in Table 2. The results showed that the polysaccharide material extracted by ammonia was between 78 and 113% of the amount recovered by direct hydrolysis of the sample. Consequently, when a paint sample is extracted with ammonia, two fractions are obtained: a lipid-resinous fraction and a polysaccharide-proteinaceous fraction. The polysaccharideproteinaceous fraction also contains soluble inorganic materials, as well as free soluble organic acids. The proteinaceous-polysaccharide fraction can be dried under a nitrogen flow and reconstituted in a TFA solution to be then subjected to ether extraction. In the ethereal phase, the free organic acids pertaining to the lipid-resinous fraction are extracted and admixed with the residue of the ammonia extraction. After partial removal of the dissolved ether under nitrogen flow, the aqueous fraction is a solution containing polysaccharides, proteins, peptides, and soluble inorganic salts. The lipid-resinous fraction admixed with the ethereal extracts can be subjected to saponification/salification, derivatization and GC/MS analysis according to the procedure described in ref 7. 2.1.3. Assessment of the Behavior of Polysaccharides Materials Subjected to the C4 Cleanup Step. The C4 stationary phase has been proposed to eliminate any interference in the analysis of proteinaceous materials from inorganic materials.7 It has been demonstrated that proteins and peptides (obtained with the ammonia extraction from solid samples) are loaded onto the stationary phase (up to the tip capacity), while inorganic materials are not. As demonstrated in section 2.1.2, after the ammonia step, a polysaccharide-proteinaceous fraction is obtained. The effect of the cleanup step on polysaccharides must thus be studied. Reference painting samples of plant gums were subjected to ammonia extraction. The extracted ammonia solution was dried under a nitrogen flow to remove ammonia, reconstituted in TFA solution, subjected to ether extraction, and, after the removal of the dissolved ether, applied onto the pipet tip containing the C4 stationary phase, using aspiration/dispensation cycles. The tip was then rinsed to remove weakly bonded compounds. Proteins and peptides loaded on the tip were subsequently eluted. Three solutions were thus generated: an eluted solution containing highly purified proteins and peptides, a residual solution, and a rinsing solution. The residual and the rinsing solutions were admixed together, dried under a nitrogen flow, hydrolyzed, and derivatized according to the procedure described in Figure 7 to determine the amount of polysaccharide materials present in each one. To summarize, the percentage of polysaccharide material eluted with the protein fraction was lower than 1% for the three gums tested, and the percentage of the saccharide material recovered from the rinsing solution admixed with the residual solution was as follows: arabic gum, 47% (RSD 14%) of the amount that is recovered after direct hydrolysis of the sample; tragacanth gum, 54% (RSD 3%) of the amount that is recovered after direct hydrolysis of the Analytical Chemistry, Vol. 82, No. 1, January 1, 2010

383

Figure 7. Optimized analytical procedure for the characterization of saccharide materials in paint samples. Table 2. Recovery of Polysaccharide Material Obtained When Subjecting the Reference Paint Layers of Polysaccharide Gum to 2.5 M Ammonia Extraction, in Comparison with Recoveries Obtained without Extractiona no extraction

NH3 extraction

gum

% recovery

RSD

% recovery

RSD

arabic tragacanth fruit tree

64 23 62

18 18 5

50 26 53

30 10 4

a

Five replicates were analyzed for each sample.

sample; and fruit tree gum, 42% (RSD 17%) of the amount that is recovered after direct hydrolysis of the sample. At this stage, it is possible to make a very rough estimation of the minimum amounts of polysaccharide binders that may be detected with this procedure. The LOD of polysaccharide material, calculated as the sum of the LODs of all the sugars determined, was 310 ng (glucose is excluded since it is not used to identify polysaccharide gums8). This indicates that analyzing with the combined analytical procedure a paint sample of 0.5 mg, with an arabic gum content which is 5% of the sample weight, leads to a signal that is about 2.5 times higher than the LOD. The procedure is thus well-suited for the characterization of saccharide binders in paint samples. The use of C4 sorbent tips enabled us to obtain two fractions. The fraction containing highly purified peptides and proteins constitutes the proteinaceous fraction. The solution (residual plus rinsing) containing polysaccharide materials, soluble inorganic materials, and the residue of the proteinaceous materials that exceeded the tip capacity, constitutes the polysaccharide fraction. The proteinaceous fraction can thus be subjected to hydrolysis, derivatization, and GC/MS analysis.7 The polysaccharide fraction can be subjected to hydrolysis, cleanup, derivatization, and GC/ MS analysis according to the procedure described in Figure 7. An analysis of the reference paint layers of the three plant gums 384


following this procedure led to chromatographic profiles that are perfectly in accordance with the literature data1,8,17 2.1.4. Remarks on the Combined Analytical Procedure. Our study highlighted that the analysis of proteinaceous, lipid, and resinous materials is not affected by the presence of polysaccharides. In addition, it also demonstrated that polysaccharide materials can be analyzed in the same paint microsample in which proteinaceous, lipid, and resinous materials are investigated. With this procedure, three fractionssa proteinaceous, a polysaccharide, and a lipid-resinous fractionsare obtained using the ammonia extraction and the C4 cleanup steps. Each fraction can be analyzed separately by GC/MS after being subjected to hydrolysis and derivatization procedures. 2.2. Analysis of Paint Samples from Works of Art. 2.2.1. 15th Century Panel Painting. The sample showed the occurrence of proteinaceous, lipid, resinous, and saccharide materials. The sum of the quantified amino acids was 11.2 µg, which is above the evaluated LOQ. The relative percentage content of quantified amino acids was as follows: Ala, 4.5; Gly, 5.0; Val, 13.5; Leu, 5.7; Ile, 8.2; Ser, 29.3; Pro, 2.0; Phe, 6.3; Asp, 10.5; Glu, 15.0; Hyp, 0.0. Figure 8 shows the PCA score plot (the paint sample is indicated with a star). The position of the sample in the PCA score plot indicates that egg is present in the sample. Figure 9 shows the TIC chromatogram relative to the lipid-resinous fraction of the paint sample. It is possible to observe the following: (1) Azelaic, myristic, sebacic, palmitic, and stearic acids point to the presence of a glycerolipid material as the main component of the lipid-resinous fraction. The ratio between the relative content of azelaic acid and palmitic acid (A/P), the ratio between the relative content of palmitic and stearic acids (P/S), and the sum of the percentage content of dicarboxylic acids (∑D) were evaluated to determine the source of the lipid material: A/P ) 0.9, P/S ) 1.1, ∑D ) 39%. The A/P and ∑D values clearly indicate that, together with egg, a drying oil was used as binder.2 The P/S ratio may be ascribed

Figure 8. PCA score plot. The paint sample is indicated with a star.

Figure 9. Chromatogram of the lipid-resinous fraction of the panel paint sample.

Figure 10. Chromatogram of the saccharide fraction. I.S. stands for internal standard: mannitol.

to linseed oil.21 P/S ) 1.1 is lower than the values commonly used as references.1 Contamination of the painting, previous restorations, the procedures used for preparing the paint media, the presence of some pigments, and the conservation conditions could all be responsible for the divergences observed from the literature values. (2) Dehydroabietic acid together with 7-oxodehydroabietic acid are the main constituents of an oxidized diterpenoid resin from the Pinaceae family. (3) Butolic, epilaccishellolic, laccishellolic, epishellolic, and shellolic acids point to the presence of an oxidized sesquiterpenoid resin of animal origin: shellac. (21) Keune, K.; Hoogland, F.; Boon, J. J.; Peggie, D.; Higgitt, C. In Prooceedings of the 15th Triennial Conference New Delhi; Allied Publishers Pvt Ltd: New Delhi, 2008; Vol. II, pp 833-842.

Figure 11. PCA score plot relative to samples from the Eastern Buddha.

Figure 12. TIC chromatogram of the saccharide fraction of sample 188-4. F1-9 are peaks ascribable to fructose.

Figure 10 shows the TIC chromatogram of the saccharide fraction. Glucose is the only sugar present above the LOD. The presence of glucose can be ascribed to a glucose-containing material, such as starch. 2.2.2. Eastern Buddha (Bamiyan, Afghanistan). The two subsamples showed the presence of nondrying fats, most likely of animal origin, characterized by the presence of (1) monocarboxylic acids with an even number of carbons (palmitic being the most abundant), (2) small amounts of dicarboxylic acids (azelaic being the most abundant), and (3) small amounts of monocarboxylic acids with an odd number of carbons. In both samples, proteinaceous material was identified at a level higher than the LOQ. The relative amino acid percentage content of the samples is reported in Table 3 together with the protein content, calculated as the sum of the 11 quantified amino acids. None of the samples presented hydroxyproline in their composition, indicating the absence of animal glue. The relative amino acid percentage content was subjected to a multivariate statistical analysis together with a data set of 121 reference samples of animal glue, egg, and casein, using principal components analysis (PCA). The resulting score plot is presented in Figure 11. The position of the samples in the score plot indicates that both samples contain egg. Since in the lipid-resinous fraction both samples showed the presence of nondrying fats of animal origin, it is possible to hypothesize that whole egg (or egg yolk) was the material used. Analytical Chemistry, Vol. 82, No. 1, January 1, 2010

385

Table 3. Relative Amino Acid Percentage Content of the Amino Acid Fraction of the Samples from the Eastern Buddha sample

Ala

Gly

Val

Leu

Ile

Ser

Pro

Phe

Asp

Glu

Hyp

protein content/µg

188-4 188-3

6.7 8.0

7.7 13.5

8.4 13.1

12.0 20.8

5.3 11.3

19.7 6.3

3.3 12.8

5.4 5.7

11.5 4.2

19.9 4.3

0.0 0.0

3.1 0.3

Table 4. Relative Sugar Percentage Content of the Saccharide Fraction of the Samples from the Eastern Buddha sample

xylose

arabinose

ramnose

fucose

galacturonic acid

glucuronic acid

glucose

mannose

galactose

saccharide content/µg

188-4 188-3

7.8 yes

13.6 yes

0.5

0.8

0.6

2.1

50.8 yes

4.0

19.8 yes

3.56 -

Regarding the saccharide fraction, sample 118-4 contained sugars above the LOQ, while sample 188-3 showed the occurrence of sugars between the LOQ and the LOD level. The glycoside profiles of the two subsamples are presented in Table 4, and the saccharide content of sample 188-4, calculated as the sum of the nine quantified sugars, is also reported. All the sugars in sample 188-4 were above the LOQ, while sample 188-3 presented only xlylose, arabinose, glucose, and galactose between the LOD and the LOQ. Considering that the samples were separated by a scalpel under the stereomicroscope, the saccharide content of sample 188-3 is most likely due to a contamination from the upper paint layer. Figure 12 presents the TIC chromatogram of the saccharide fraction of sample 188-4. The presence of all aldoses and uronic acids and the high content of glucose and fructose indicate that the saccharide material present is not arabic, tragacanth, or fruit three gum but must be ascribed to a different saccharide material. The saccharide profile observed was not related to any of those reported in the literature, but it is important to stress that the main sources of fructose are fruit, vegetable, and honey. Most likely, the material used was a mixture of saccharide materials locally available to the artist. The discussion of the painting technique is quite complex, requiring a systematic campaign and the support of conservators to better understand the aesthetic function of the various paint layers and their originality, but this is beyond the scope of this paper. Nevertheless, it is possible to assert that the white layer (sample 188-3) was painted using egg as a binder. On top of this layer, a yellow layer (sample 188-4) was applied using a mixture of egg and a saccharide binder. 3. CONCLUSIONS A combined analytical procedure for the simultaneous characterization of drying oils, animal and plant terpenoid resinous materials, natural waxes, polysaccharide and proteinaceous materials on the same microsample using GC/MS was set up. The procedure is also reliable when high amounts of inorganic materials are present. A lipid-resinous, a proteinaceous, and a saccharide fraction, to be analyzed separately, were all obtained from the same sample, using ammonia extraction and a C4 stationary phase placed on a pipet tip. The procedure was successfully used to characterize the organic materials in a sample from a panel painting from the 15th

386


century, and one from the Eastern Buddha (Bamiyan, Afghanistan) from the 6th century. The analyses on the first painting enabled us to highlight the potential of the analytical procedure to simultaneously characterize proteinaceous, resinous, lipid, and polysaccharide materials in the same sample. The differential sampling of the paint layers performed on the sample collected from the Eastern Buddha showed how this procedure can be used to understand the organic material composition in the sample buildup. The results highlighted that the analytical procedure presented is suitable for the determination of the polysaccharide binders in the same sample used to determine proteinaceous, glycerolipids, waxy, and resinous materials. This is extremely important when unique and, often, physically indivisible samples from valuable works of art are available. When the sample can be divided into different fractions to be analyzed using different analytical procedures, there is always a risk of losing information due to sample losses and contamination. In addition, being able to determine all the organic materials simultaneously is fundamental when subsamples are obtained from the original sample (for example, when separating different paint layers) or when highly heterogeneous samples are available. In fact, only in this way is it possible to be sure that the materials determined belong to the same subsample and do not relate to different areas or layers of the same sample. ACKNOWLEDGMENT The authors gratefully acknowledge the contributions of Prof. Michael Petzet, former World president of ICOMOS; Roland Lin, UNESCO World Heritage Centre; The Afghan Ministry of Culture, Department of Historical Monuments MoIC and Ministry of Urban Development; Edmund Melzl, who collected the fragments of the Bamiyan Buddhas; Prof. Erwin Emmerling; Catharina Bla¨nsdorf and Stephanie Pfeffer of the Department for Restoration, Art Technology and Conservation Science, Technical University Munich, who were in charge of the fragment investigation. Rita Carosi, of the Department of Chemistry and Industrial Chemistry of University of Pisa is acknowledged for the FTIR analyses.

Received for review September 23, 2009. Accepted November 6, 2009. AC902141M

MS Analytical Procedure for the Characterization of Glycerolipids

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