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Identification of Organic Materials in Historic Oil Paintings Using Correlated Extractionless Surface-Enhanced Raman Scattering and Fluorescence Microscopy Lindsay H. Oakley,† Stephen A. Dinehart,† Shelley A. Svoboda,‡ and Kristin L. Wustholz*,† † ‡
Department of Chemistry, The College of William and Mary, Williamsburg, Virginia 23187, United States Department of Conservation, The Colonial Williamsburg Foundation, Williamsburg, Virginia, 23187, United States ABSTRACT: A novel spectroscopic approach, correlated surface-enhanced Raman scattering (SERS) and fluorescence microscopy, is used to identify organic materials in two 18th century oil paintings. The vibrational fingerprint of analyte molecules is revealed using SERS, and corresponding fluorescence measurements provide a probe of local environment as well as an inherent capability to verify material identification. Correlated SERS and fluorescence measurements are performed directly on single pigment particles obtained from historic oil paintings with Ag colloids as the enhancing substrate. We demonstrate the first extractionless nonhydrolysis SERS study of oil paint as well as the potential of correlated SERS and fluorescence microscopy studies for the simultaneous identification of organic colorants and binding media in historic oil paintings.
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he analysis of natural, organic colorants in historical artworks is one of the most challenging tasks in conservation science. Conventional analytical techniques such as UV/vis spectrophotometry,1 fluorimetry,24 high-performance liquid chromatography (HPLC),1,5,6 and Raman spectroscopy7,8 have significant disadvantages for the study of organic-based artists’ materials that include a large sample requirement, poor sensitivity, poor selectivity, and the inability to unambiguously identify many organic colorants. For example, although Raman spectroscopy measures the unique vibrational fingerprint of analytes, typical Raman cross sections are small and strong molecular fluorescence from natural dyestuffs often precludes the measurement of Raman scattering.9 To circumvent the problems associated with traditional analytical approaches, recent studies have focused on the application of surface-enhanced Raman scattering (SERS) to the identification of organic colorants in works of art.5,1019 The noble-metal SERS substrate not only provides enhanced Raman signals such that minute sample sizes (i.e., single pigment grains) are measurable but also quenches the fluorescence generated by many organic colorants. For example, Brosseau et al. have applied extractionless nonhydrolysis SERS to the detection of red lake pigments in pastels and watercolors.16 In these experiments, Ag nanoparticles were applied directly to samples and the pigments were identified without the need to extract or hydrolyze the colorant from relatively simple, homogeneous matrixes. Despite these successes, the widespread application of SERS to the study of historic artworks has proven somewhat challenging. First, extractionless nonhydrolysis SERS has not been successfully applied to the study of organic colorants in complex, heterogeneous matrixes such as oil paints and glazes. Historic r 2011 American Chemical Society
oil paintings may contain an amalgamation of colorants, resins, gums, waxes, and oils, all of which have undergone aging and degradation. Due to this inherent complexity, samples are often pretreated prior to investigation using SERS. Indeed, Leona and co-workers demonstrated the only SERS study of oil glazes, wherein hydrofluoric acid was used to pretreat a sample in order to extract the colorant from the binding medium.13 Although pretreatment with HF is an effective approach, a direct extractionless nonhydrolysis analysis of samples from oil paintings is preferable in order to (1) preserve the integrity of the sample, (2) remove the pretreatment step, and (3) eliminate the hazards associated with the use of HF. Another challenge in applying SERS to the study of artwork is that little information about the environment of the colorant is elucidated. In particular, SERS is limited in its ability to detect nonresonant materials (e.g., oils, resins) that coexist with organic colorants, owing to the strong resonance Raman effect of chromophores that contributes to the overall SERS enhancement.20 The development of a reliable, comprehensive SERS-based method to study artists’ materials requires overcoming these challenges. Here, we develop a novel approach for the study of organic materials in historic oil paintings using correlated extractionless nonhydrolysis SERS and fluorescence microscopy. This coupling provides a uniquely sensitive and specific spectroscopic tool for conservation science that is able to measure both the electronic and vibrational properties of colorants. Furthermore, fluorescence measurements provide a probe of local environment as Received: March 18, 2011 Accepted: April 28, 2011 Published: April 28, 2011 3986
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Analytical Chemistry
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Figure 1. Correlated SERS and fluorescence analysis of the (a) Portrait of William Nelson by Robert Feke, probably 17481750. (b) Photomicrograph of lip sample coated in 0.75 μL of Ag colloidal paste. (c) Corresponding SERS spectrum of the sample obtained at 632.8 nm, with Pexc = 40 μW and taq= 90 s. Labeled peaks are characteristic of carmine lake and linseed oil, and the asterisks denote bands due to citrate.15,16 (d) Fluorescence spectra of the lip sample (solid line) and a reference paint of carmine lake in linseed oil (dashed line).
well as inherent capability to verify material identification. Two oil paintings from the Colonial Williamsburg Foundation collection are analyzed. The first, Portrait of William Nelson (Figure 1a) by Robert Feke, is a painting by the earliest native-born American artist of European descent. In order to highlight the unique advantages of this approach, we investigated a second painting that was likely to demonstrate exceptional complexity, a portrait by Sir Joshua Reynolds, a founder of the Royal Academy of Arts. Reynolds undertook complex experimentation with organic binding and coating materials.21 In both paintings, portions of the fleshtones appear slightly faded, suggesting the presence of organic red lake pigments. Ultimately, this work demonstrates the first extractionless nonhydrolysis SERS study of organic materials in oil paintings and the utility of correlated SERS and fluorescence microscopy to the study of historical artworks.
’ EXPERIMENTAL SECTION Materials, Synthesis, and Sample Preparation. Carmine naccarat (aluminum lake of carminic acid), madder lake, burnt sienna, walnut oil, linseed oil, Cremnitz white, and lamp black were obtained from Kremer Pigments. Reference paints (pigment with linseed oil), tints (paint with Cremnitz white), and tones (tint with lamp black and burnt sienna) of carmine lake were prepared from these materials using historic methods and applied to glass slides (Fisher). Microscopic samples were obtained from oil paintings with surgical blades (Feather Safety Razor Company). Samples from Portrait of William Nelson (by Robert Feke, oil on canvas, probably 17481750, 50-5/8 40-5/8 in.; CWF 1986-245) and Portrait of Isaac Barre (by Sir
Joshua Reynolds, oil on canvas, 1766, 50-1/16 40-1/16 in.; CWF 2010-103) were investigated. Glassware was cleaned with aqua regia prior to use. Silver nitrate (Acros Organic, 99%þ) and sodium citrate (Sigma Aldrich) were used to synthesize citrate-reduced Ag colloids,22 resulting in an opaque, gray-green solution. The colloids were centrifuged (Eppendorf, MiniSpin, 1 mL aliquots with ∼0.9 mL of supernatant removed) for two cycles at a relative centrifugal force of ∼12 000 g at 15 min per cycle. The microscopic art samples were placed on clean glass coverslips and coated in 0.75 μL of Ag colloids. Cross-Section Analysis. Cross sections were mounted in polyester resin (Ward’s Bio-Plastic), polished (Micro-Mesh, Inc., 12,000 grit), examined with a microscope (Nikon Eclipse E600) under white-light (OPELCO, fiber-optic halogen source) and UV illumination (100 W Hg lamp) at 200 magnification, and imaged with a camera (Nikon D80). SERS and Fluorescence Measurements. Correlated SERS and fluorescence measurements were performed on an inverted microscope (Nikon, TiU) coupled to a 1/3 m imaging spectrograph (Princeton Instruments, SP2356) equipped with a CCD camera (Princeton Instruments, PIXIS:100B-Excelon). For normal Raman and SERS measurements, excitation at 632.8 nm from a HeNe laser (Research Electro-Optics, LHRP-1701) was filtered (Semrock, LL01-633-25) and focused to the sample using a 20 objective (Nikon CFI, N.A. = 0.5). Scattering from the sample was collected through the objective, filtered (Semrock, LP02-633RS-25), and focused to the entrance slit of the spectrograph. Raman scattering was dispersed using a 600 g/mm grating blazed at 500 nm. The observed Raman frequencies were 3987
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Analytical Chemistry determined by calibrating to a cyclohexane standard. Typical excitation power (Pexc) and acquisition time (taq) for SERS measurements were set to ∼40 μW and 60 s, respectively, in order to maximize signal-to-noise and prevent molecular photobleaching. Fluorescence measurements were performed on the inverted microscope using 532 nm excitation (Spectra Physics, Excelsior) that was filtered (Chroma, z532/633x) and focused to the sample with a 20 objective. Epi-fluorescence was collected through the objective, filtered (Semrock, LP03-532RS-25), and
Figure 2. (a) Portrait of Isaac Barre by Sir Joshua Reynolds, 1766. (b) Photomicrograph of cheek, with a lake pigment particle highlighted. Cross section of red paint imaged in (c) white and (d) UV light. A = preparation layer, B = paint layers containing red lake pigments, and C = varnish.
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dispersed with a 150 g/mm grating. Typical Pexc and taq for fluorescence measurements were 8 μW and 10 s, respectively. A color camera (Edmund Optics, EO-0413C) was used to record images of the samples. Emission from reference samples was measured in the presence and absence of colloids; the spectra exhibited modest differences in intensity but not wavelength.
’ RESULTS AND DISCUSSION A photomicrograph of a single pigment grain obtained from the lip region of the Portrait of William Nelson and coated in Ag colloids is presented in Figure 1b. The colloidal paste is not entirely adhered to the sample, which is likely due to the hydrophobicity of the oil medium. Owing to this heterogeneous coverage, we observed that some regions of the sample exhibited excellent SERS signal while others produced none (i.e., fluorescence only). Figure 1c presents the SERS spectrum of the sample obtained at 632.8 nm excitation. Major peaks are observed at 1483 cm1 (m), 1463 cm1 (m), 1436 cm1 (m), 1300 cm1 (s), 1264 cm1 (sh), 1107 cm1 (w), 1088 cm1 (w), 672 cm1 (w), 471 cm1 (m), and 434 cm1 (m), indicating the presence of carmine lake, consistent with previous Raman and SERS studies.15,16,23 A modest peak at 875 cm1 suggests the presence of linseed oil,24 but this observation alone is insufficient for reliable identification. The corresponding fluorescence spectrum of the sample following 532 nm excitation exhibits an emission maximum at 634 nm (Figure 1d). Emission from aqueous solutions of carminic acid, the principal dye component in carmine lake, demonstrate a maximum at 586 nm. However, the fluorescence of carminic acid is solvent dependent.25,26 Indeed, the sample fluorescence spectrum is an excellent match
Figure 3. Correlated extractionless nonhydrolysis SERS and fluorescence measurements of samples from the Portrait of Isaac Barre. SERS spectra of single pigment grains from the (a,b) cheek and (c) finger obtained at 632.8 nm using Pexc ≈ 40 μW and taq= 60 s. Labeled peaks are consistent with carmine lake, and asterisks denote peaks due to citrate.15,16 (d) Emission spectra of reference carmine-lake-based: paint (solid, pigment and linseed oil), tint (dashed, paint with Cremnitz white), and tone (dashed-dotted, tint with burnt sienna and lamp black). (e) Corresponding fluorescence from single pigment grains taken from the cheek (solid, dashed) and finger (dashed-dotted) demonstrate two maxima. The sharp cutoff at ∼550 nm is from the emission filter. 3988
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Analytical Chemistry to a reference paint composed of carmine lake and linseed oil (Figure 1d).26 The Portrait of Isaac Barre (Figure 2a) by Sir Joshua Reynolds demonstrates exceptional material complexity. Figure 2b shows a photomicrograph of the painting’s surface. Figure 2c,d presents a cross section of red paint imaged in white and UV light, respectively. The cross-section analysis reveals a preparation layer, paint layers containing red lake pigments as evidenced by fluorescence under UV illumination, and a varnish layer. Representative SERS spectra of single pigment grains taken from the Portrait of Isaac Barre are shown in Figures 3ac. Samples from the cheek and finger flesh exhibit SERS bands consistent with carmine lake and linseed oil (i.e., ∼875 cm1 in Figure 3c),24 as well as modest peaks (i.e., 892 cm1 and 772 cm1) attributed to nonresonant species. To investigate the composition of the surrounding environment, fluorescence spectra of the samples were measured and compared to knowns. The fluorescence spectra of the reference carmine-lake-based paint, tint, and tone exhibit maxima at 634 ( 5 nm (Figure 3d), indicating a relative insensitivity to the surrounding matrix (e.g., with the addition of nonresonant colorants such as Cremnitz white). In contrast, two broad fluorescence peaks at ∼598 and 634 nm are observed for portrait samples (Figure 3e), suggesting that the matrix contains linseed oil as well as additional fluorescent species. Fluorescent terpenoid resins27 are consistent with the materials knowledge of the artist and time period.21 In particular, copal exhibits broad fluorescence >500 nm that is dependent on excitation wavelength28 and is significantly red-shifted upon aging,29,30 as well as vibrational bands at 889 cm1 and 772 cm1.31 Other prominent Raman bands for copal at ∼1650 cm1 and ∼1400 cm1 may be obscured by resonance Raman signal of the chromophore. Therefore, our observations indicate that Reynolds used carmine lake, linseed oil, and copal in the fleshtones of the Portrait of Isaac Barre. Correlated extractionless nonhydrolysis SERS and fluorescence microscopy proved to be a powerful tool for the study of historic oil paintings. Using this approach, an exceptionally small sample is adequate to provide definitive identification of the organic colorant, without the need for sample pretreatment. Furthermore, our preliminary results demonstrate the potential of this correlated method for the simultaneous identification of organic colorants and binding media in works of art. In total, 37 out of 42 single grains taken from various regions of historic oil paintings exhibited excellent correlated SERS and fluorescence spectra. These results highlight the need for further mechanistic studies of pigmentnanoparticle interactions and spectral libraries of known and deliberately aged paint samples. Future work will focus on the study of additional organic materials and their degradation pathways.
’ AUTHOR INFORMATION Corresponding Author
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*Fax: (757) 221-2715. E-mail:
[email protected].
’ ACKNOWLEDGMENT We thank the College of William and Mary for support through startup funds to K.L.W. and Prof. Christa Brosseau of St. Mary’s University for helpful discussions. Painting images are courtesy of the Colonial Williamsburg Foundation. 3989
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