Anal. Chem. 2007, 79, 6143-6151
Raman Spectra of Proteinaceous Materials Used in Paintings: A Multivariate Analytical Approach for Classification and Identification Austin Nevin,*,†,‡ Iacopo Osticioli,§ Demetrios Anglos,† Aviva Burnstock,‡ Sharon Cather,‡ and Emilio Castellucci§,⊥
Department of Chemistry, University of Florence, Polo Scientifico e Tecnologico, via della Lastruccia 3, I-50019, Firenze, Italy, Institute of Electronic Structure and Laser, Foundation for Research and Technology Hellas (IESL-FORTH), P.O. Box 1385, Heraklion, 71110, Greece, Courtauld Institute of Art, Somerset House, Strand, London, WC2R 0RN, London, U.K., and European Laboratory for Nonlinear Spectroscopy (LENS), University of Florence, Polo Scientifico e Tecnologico, Via Nello Carrara 1, I-50019 Firenze, Italy
This work presents Raman spectra obtained from thin films of protein materials which are commonly used as binding media in painted works of art. Spectra were recorded over the spectral range of 3250-250 cm-1, using an excitation wavelength of 785 nm, and several bands have been identified in the fingerprint region that correspond to the various proteins examined. Differences in the C-H vibrations located between 3200 and 2700 cm-1 can be accounted for with reference to the amino acid composition of the protein-based binding media as well as the presence of fatty acid esters, in the case of egg yolk. In addition, the discrimination of different proteins on the basis of variations in spectra between 3200 and 2700 cm-1 can be achieved following multivariate analysis of a large data set of spectra, providing a novel and nondestructive alternative based on Raman spectroscopy to other methods commonly used for the analysis of proteins. Protein-based materials from egg white and egg yolk, milk and casein, and animal glues have traditionally been used as binding media for paintings, in restoration and treatments, and thus their identification is critical for conservation. Due to the inherent adhesive properties of commonly found food-based proteins (from eggs, milk, and cheese), and the ease with which animal glues (from hides, bones, and skins) can be extracted, it may not be surprising that early treatises on painting and the arts, ranging from Pliny to Cennino Cennini1 refer to the use of proteins for paint. Wall paintings, easel paintings, and sculptures have often been executed or decorated using protein-based binding media, and this is due to the flexibility and versatility which such media provide; proteins are compatible with pigments, they are durable and relatively stable. In addition to the use of proteins as original * To whom correspondence should be addressed. E-mail: austin.nevin@ courtauld.ac.uk. † IESL-FORTH. ‡ Courtauld Institute of Art. § Department of Chemistry, University of Florence. ⊥ LENS. (1) Cennini, C. d. Il libro dell’Arte; Dover Publications: New York, 1933. 10.1021/ac070373j CCC: $37.00 Published on Web 07/10/2007
© 2007 American Chemical Society
materials, conservation treatments often make use of similar adhesives for fixing flakes on painted surfaces, thus complicating the determination of the original and added materials found in works of art. Not only for historic interest, but also for the development of responsible and careful conservation interventions which do not compromise the integrity of the original material, the characterization of painting materials, which includes the binding medium and not only the pigments found, is crucial to the conservation of art. Most paintings consist of multiple layers of inorganic and/or organic pigments and colorants which are typically dispersed in a matrix applied on a substrate and are often coated with an organic varnish. Proteinaceous materials found in paintings are not generally pure materials but rather contain mixtures of different proteins as well as fatty acid esters (for example egg yolk), various vitamins, and impurities related to preparation, aging, and deterioration processes. The analysis of proteins and other binding media typically involves taking samples. Reliable and specific information regarding proteinaceous materials can be obtained from the analysis of small samples by means of sensitive techniques, such as liquid chromatography2 and gas chromatography-mass spectrometry (GC/MS),3 for which various protocols have been established4 based on multivariate analysis of the amino acid composition of unknown samples compared to a reference database. These methods often lead to precise and quantitative identification of binding media but require sampling and elaborate preparation, often including hydrolytic treatment and derivatization. Although characterization using noninvasive methods is clearly advantageous, few optical techniques are available for the analysis of proteins and include portable near-infrared fiber-optic reflectance spectroscopy (FORS)5 and laser-induced fluorescence (LIF) (2) Grzywacz, C. M. J. Chromatogr., A 1994, 676, 177-183. (3) Andreotti, A.; Bonaduce, I.; Colombini, M. P.; Gautier, G.; Modugno, F.; Ribechini, E. Anal. Chem. 2006, 78, 4490-4500. (4) Colombini, M. P.; Modugno, F. J. Sep. Sci. 2004, 27, 147-160. (5) Fabbri, M.; Picollo, M.; Porcinai, S.; Bacci, M. Appl. Spectrosc. 2001, 55, 428-433.
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spectroscopy;6 however, broad spectral features reduce the potential of these methods to discriminate among similar materials. In situ analysis, which requires no sampling, is particularly desirable because there is no limit to the number of measurements and it does not require the expensive and difficult transport of an object to a laboratory, but results can be compromised by a lack of specificity, resolution, and sensitivity, or due to interferences from inorganic materials. In FT-IR spectroscopy a sample is required but not necessarily destroyed during analysis, and FT-IR is commonly used for the analysis of both inorganic and organic materials found in paintings, including proteins. The determination of the presence of proteinaceous material on the basis of characteristic vibrational bands7 is sometimes possible,8 but the identification of a specific binding medium is not generally feasible. However, other types of proteinbased materials, including solid tortoiseshell and its imitation galalith, can be discriminated using attenuated total reflectance (ATR) FT-IR combined with spectral deconvolution and correlation.9 Further, it should be noted here that considerable improvements have been achieved through applications of synchrotron FT-IR due to improved spatial resolution and sharper bands.10 In Raman spectroscopy, which is the focus of this article, sharper and better-resolved vibrational bands can be obtained from the analysis of both organic and inorganic materials. Additionally, micro-Raman spectroscopy can be used to analyze samples smaller than 5 µm × 5 µm; thus, it often permits the selective analysis of the organic (binding medium) component of a paint, hence reducing the interferences from adjacent pigment particles. In contrast to micro-FT-IR,11 micro-Raman provides improved selectivity and specificity which can be particularly useful for the determination of composition. Micro-Raman is commonly associated with the determination of inorganic pigments,12-15 and the analysis of archeological remains such as degraded collagen16 and solid protein materials including tortoiseshell17 has been carried out using Fourier transform Raman spectroscopy. Raman spectroscopy has seldom been applied to the analysis of organic materials found in paintings,13,18,19 and proteinaceous binding media have rarely been considered.19 This is due to the masking of Raman scattering by fluorescence which is intrinsic (6) Nevin, A.; Cather, S.; Anglos, D.; Fotakis, C. Anal. Chim. Acta 2006, 573, 341-346. (7) Mikhonin, A. V.; Ahmed, Z.; Ianoul, A.; Asher, S. A. J. Phys. Chem. B 2004, 108, 19020-19028. (8) Pilc, J. W. R. National Gallery Technical Bulletin 1995, 16, 73-95. (9) Paris, C.; Lecomte, S.; Coupry, C. Spectrochim. Acta, Part A 2005, 62, 532538. (10) Salvado, N.; Buti, S.; Tobin, M. J.; Pantos, E.; Prag, A. J. N. W.; Pradell, T. Anal. Chem. 2005, 77, 3444-3451. (11) van der Weerd, J.; van Veen, M.; Heeren, R.; Boon, J. Anal. Chem. 2003, 75, 716-722. (12) Bell, I. M.; Clark, R. J. H.; Gibbs, P. J. Spectrochim. Acta, Part A 1997, 53, 2159-2179. (13) Burgio, L.; Clark, R. J. H. Spectrochim. Acta, Part A 2001, 57, 1491-1521. (14) Anglos, D. Lasers for the Preservation of Cultural Heritage: Principals and Applications; Fotakis, C., Anglos, D., Georgiou, S., Zaphiropolous, V., Tornari, V., Eds; Francis and Taylor, 2006. (15) Smith, G.; Clark, R. Reviews in Conservation 2002, 2, 92-106. (16) Williams, A. C.; Edwards, H. G. M.; Barry, B. W. Biochim. Biophys. Acta 1995, 1246, 98-105. (17) Paris, C.; Coupry, C. J. Raman Spectrosc. 2005, 36, 77-82. (18) Burrafato, G.; Calabrese, M.; Cosentino, A.; Gueli, A. M.; Troja, S. O.; Zuccarello, A. J. Raman Spectrosc. 2004, 35, 879-886. (19) Vandenabeele, P.; Wehling, B.; Moens, L.; Edwards, H.; De Reu, M.; Van Hooydonk, G. Anal. Chim. Acta 2000, 407, 261-274.
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to protein-based binding media,6 a common problem in the application of micro-Raman for the analysis of both inorganic and organic materials. However, recent improvements in instrumentation (which include near-IR excitation with dispersion or FT-Raman instruments, gated Raman, and anti-Stokes Raman) as well as the application of mathematical methods are promising for overcoming the problem of fluorescence.20,21 The lack of extensive spectral databases of reference materials and the time required for spectral collection are further complications for the use of Raman spectroscopy for the analysis of binding media. However, Raman spectroscopy has been used for the analysis of solid protein-based compounds found in food which include lysozyme (egg white),22 lactalbumin (milk),23 casein,24 cheese,25,26 and animal gels.27 The assignment of spectra to specific molecular vibrations is problematic for macromolecules, but research on analogous samples26,28 is particularly useful for interpretation of Raman spectra of protein-based binding media. For the identification of protein-based binding media, micro-Raman results suggest that indicative vibrations are analogous to many of those found in FTIR: amide I (approximately 1670 cm-1), CH2 scissoring (approximately 1250 cm-1), and amide III (approximately 1250 cm-1).19 However, vibrations from C-H stretching between 3200 and 2800 cm-1 (using visible excitation) have not been reported for protein-based binding media. In addition to the assignment of Raman spectra, multivariate analysis, recently employed in biological applications of Raman spectroscopy for cellular discrimination,29 has also been used to identify small differences between synthetic and natural organic pigments, based on small variations on spectral shape and peak position.30 Although principal component analysis (PCA) is routinely applied for the analysis of amino acid composition for the determination of binding media following GC/MS,3 multivariate analysis has not been applied for the discrimination of proteinaceous binding media on the basis of their Raman spectra. PCA, also known as eigenvector analysis, can be used to explain the variance-covariance structure of a set of data by using a linear combination of original variables to form principal components (PC). Combinations of the original dimensions describe the largest variance between data sets, and most of the information from the original data set can be accounted for by a smaller number of variables. This work systematically investigates the potential of Raman spectroscopy to identify proteinaceous materials which are used as binding media in works of art including easel paintings, wall paintings, and other polychromy. A series of Raman spectra in the range from 3500 to 200 cm-1 have been recorded of films of (20) O’Grady, A.; Dennis, D.; Denvir, D.; McGarvey, J.; Bell, S. Anal. Chem. 2001, 73, 2058-2065. (21) Osticioli, I.; Zoppi, A.; Castelucci, E. M. J. Raman Spectrosc. 2006, 37, 974980. (22) Tuma, R. J. Raman Spectrosc. 2005, 36, 307-319. (23) Howell, N. K.; Arteaga, G.; Nakai, S.; Li-Chan, E. C. Y. J. Agric. Food Chem. 1999, 47, 924-933. (24) Byler, D.; Susi, H. J. Ind. Microbiol. 1988, 3, 73-88. (25) Fontecha, J.; Bellanato, J.; Juarez, M. J. Dairy Sci. 1993, 76, 3303-3309. (26) Susi, H.; Byler, D. Appl. Spectrosc. 1988, 42, 819-826. (27) Bouraoui, M.; Nakai, S.; Li-Chan, E. Food Res. Int. 1997, 30, 65-72. (28) Li-Chan, E. C. Y. Trends Food Sci. Technol. 1996, 7, 361-370. (29) Chan, J. W.; Taylor, D. S.; Zwerdling, T.; Lane, S. M.; Ihara, K.; Huser, T. Biophys. J. 2006, 90, 648-656. (30) Vandenabeele, P.; Moens, L. Analyst 2003, 128, 187-193.
eight different proteinaceous binding media (egg white, egg yolk, casein, milk, fish glue (isinglass), rabbit skin glue, ox bone glue, and parchment glue), prepared using traditional recipes.1 Spectra are interpreted with specific reference to the amino acid composition of the binding media31 as well as published Raman spectra of organic materials used as binding media13,19 and from spectra of analogous food samples.23,25 Further, PCA has been used for the first time in an effort to discriminate proteinaceous binding media on the basis of Raman bands associated with the C-H stretching vibrations. MATERIALS AND METHODS Eight representative proteinaceous binding media were chosen for this study, each of which contains different proteins: ox glue, rabbit skin glue, parchment glue, and fish glue (collagen), egg white (predominantly lysozyme and albumin), egg yolk (lysozyme, ovalbumin, as well as 40% fatty acid esters), milk (casein and lactalbumin), and extracted casein. Samples of binding media were chosen to be similar to those documented in artists’ accounts and historical recipes. Ox glue and rabbit skin glue (Kremer Pigments) were swollen and dissolved in a warm water bath in nanopure water to give a solution of 5% w/w. Parchment glue and fish glue were prepared as described by Cennini:1 parchment (dried sheep skin) and dried sturgeon bladder were cut into fine strips, and these were washed in hot water and then left to swell in water for 24 h (original concentration 5% by weight); the mixtures were then heated for 6 h at 90 °C in covered beakers, which yielded a transparent colorless solution of partially dissolved collagen (glue). Eggs from corn-fed hens were bought from a local market. Egg white was beaten to form stiff peaks and left for 24 h; foam was skimmed and removed, and a solution of 50% w/w clear egg white in nanopure water was prepared. Egg yolk was extracted from egg by piercing the yolk and allowing the liquid to drip from the encasing film; the yolk was diluted in nanopure water to give a 50% w/w emulsion. Unfortified pasteurized skimmed milk was purchased from a local market. Casein (Kremer Pigments) was swollen in water for 24 h to give a 1.5% w/w solution; dilute ammonia solution was added until the swollen gel dissolved, and excess ammonia was left to evaporate. Films of proteins were cast using prepared solutions on polished fused-silica disks to give film thickness of approximately 15 µm (measured on a Perthometer S5P stylus profilometer) and were examined 1 year following their preparation. In addition, pigmented samples were prepared using egg white mixed with yellow ochre painted out on quartz disks. Analysis was performed using a Renishaw 2000 micro-Raman spectrometer equipped with a 785 nm diode laser, and a power of 6 mW was incident on the sample during analysis. Although another laser operating at 514.5 nm is available, analysis using higher energy radiation results in a much higher luminescence background. The laser was focused onto the sample using a 50× objective, and an area of approximately 6 µm diameter was analyzed at a spectral resolution of 4 cm-1. Spectra were recorded (31) Colombini, M. P.; Modugno, F.; Giacomelli, A. J. Chromatogr., A 1999, 846, 101-111. (32) Tobin, M. Laser Raman Spectroscopy; Wiley-Interscience, John Wiley and Sons: New York, 1971. (33) Liang, M.; Chen, V. Y. T.; Chen, H.-L.; Chen, W. Talanta 2006, 69, 12691277. (34) Zhao, Y.; Ma, C.-Y.; Yuen, S.-N.; Phillips, D. L. J. Food Sci. 2004, 69, 206213.
with two different spectral ranges: the first scan was between 3500 and 250 cm-1, and the second scan recorded between 3200 and 2700 cm-1. For each scan three acquisitions at 100 s were recorded with a resolution of approximately 0.6 cm-1. The spectrometer was calibrated using the Raman signal of silicon at 520 cm-1 at the beginning and end of each day of measurements; variations fell within the spectral resolution of the instrument. Cosmic noise and spikes were removed from spectra which were corrected for detector efficiency, but otherwise no corrections (for example manual baseline adjustment) were applied. No changes in spectra were observed that could be accounted for by sample degradation or damage during spectra acquisition. It is of concern that damage to samples can be induced due to long exposure using high-intensity laser sources to small samples from paintings, equally relevant for the analysis of inorganic13 and organic materials.26 Multivariate analysis of spectra was performed on spectra collected in the range between 3200 and 2700 cm-1. A total of 80 spectra were recorded, at 10 different positions and on 10 different days for each sample. Data was interpolated to yield a spacing of 2 cm-1 between each point, giving 251 points in the selected range, which was necessary because of changes in the exact values of Raman shift due to instrumental calibration, the position of which varied from day to day. Spectra were vector normalized as described by Vandenabeele and Moens30 to account for variations in measurement geometry which may influence the overall recorded intensity. From every position in the spectrum the average intensity over all band positions in the spectrum was subtracted and then divided by the standard deviation of the peak intensities. Spectra were then compared with PCA using Software for Chemometric Analysis (SCAN), Minitab, to give principal components for each spectrum and loading plots of each component. RESULTS Raman spectra were recorded from the surface of protein films and are reported in Figure 1 in the full range; importantly, a significant fluorescence background is observed in the nonzero baseline for every protein, which is particularly pronounced in casein (Figure 1b). Although amino acids have been implicated in the fluorescence of binding media, this is observed primarily following UV excitation. The nonzero baseline in the Raman spectra from proteins observed with excitation at 785 nm can be most likely accounted for by luminescence arising from impurities commonly present in samples from natural sources. Two different regions of vibrations are characteristic of each protein. The first corresponds to the fingerprint region below 1800 cm-1, and the second corresponds to the range above 2700 cm-1 where C-H stretching is observed. Importantly for the proteins considered, although the relative intensity of the Raman signal compared to the baseline is different, vibrations from amide I at approximately 1660 cm-1 and amide III are observed in every sample, as are other peaks which are due to the presence of specific amino acids, as outlined in Table 1. Egg yolk, due to the large presence of fatty acid esters, has a characteristic carbonyl vibration at 1740 cm-1, and the C-H stretching region is also obviously different from that of the other proteins, which can be rationalized with reference to the proportion of unsaturated and saturated C-C bonds present in the film. Importantly for a consideration of Raman Analytical Chemistry, Vol. 79, No. 16, August 15, 2007
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Figure 1. Raman spectra of binding media shown in the range of 3250-400 cm-1, normalized to the amide-I stretching at approximately 1660 cm-1; excitation, 785 nm: (a) egg yolk, (b) casein, (c) egg white, (d) milk, (e) fish glue, (f) parchment glue, (g) ox bone glue, (h) rabbit skin glue.
spectroscopy for the analysis of proteins using 785 nm excitation, is that spectra of collagen-based samples are significantly less intense than those associated with the other protein-based binding media, a fact which remains to be explained. 6146
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A closer examination of the spectra in the fingerprint region, shown in Figure 2, indicates that different features may be specific to particular proteins, and an assignment of bands is given in Table 1. Although small variations in the peak position of the
Table 1. Tentative Peak Assignment of Raman Spectra Obtained from Thin Films of the Eight Different Protein-Based Media Examineda dairy proteins (Raman shift, cm-1)
tentative assignmentb
egg yolk
egg white
casein
milk
fish glue
parchment glue
rabbit glue
ox glue
605
602
604
601
600
598
603
598
644 718
698
632 703
729
739
767
758
781 816
781 814
797 815
789 813
856
857
853
852
919 938
919 921
919 938
918 938
1003
1003
1003
1002
1033
1033
1034
1033
763 780
828 849 873
1003
1080 1125
1440 1555 1657 1725 2726 2852
b
collagen (Raman shift, cm-1)
826 852 878
900 934 954
931 954
1003
1003
1032 1041
1033
1125
1127
828 851 876 915 954 1019 1032 1052 1087 1120 1140
1207 1241 1336
1206 1248 1338
1401
1410
1261 1338 1379 1414
1448 1550 1605 1660
1449 1551 1616 1665
1452 1551 1608 1663
2729 2873 3061
2870
2725 2887 2976.5
1092 1122 1206 1244 1318
1204 1242 1319
1412
1411
1449
1448 1557 1603 1665
1607 1665 2882
2880 2985.3
1165
1168
1208 1249 1315 1379
1205 1244 1313
1448 1603 1666
1448 1550 1602 1663
2873
2880
amide VI N-H deformation Tyr C-S stretching Cysteine/ trans and gauche Trp N-H wagging C-C stretching Tyr Tyr Trp C-C vibration C-C stretching C-C vibration, phosphate symmetric stretching Phe ring breathing C-C stretching Phe C-C stretching C-C, C-N stretch C-CT stretch Aliphatic CH3anti symmetric; Aromatic CH2 rock Phe/Tyr amide III CH2 deformation Aspartic and glutamic acids (C)O stretching) C-H bending Trp Phe/Tyr amide I CdO stretching aliphatic C-H stretching C-H stretching aromatic CH stretching
(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28)
a Phe ) phenylalanine, Tyr ) tyrosine, Trp ) tryptophan. Numbers in parentheses have been used to label selected peaks in Figures 2 and 3. Refs 7, 17, 19, 22, 23, 25, 28, 32-36. Bands which have not been identified are not assigned but are listed for completeness.
amide-I and amide-III vibrations have been linked to secondary structure, it is not likely that shifts in peak position in these samples can be ascribed to a predominance of random coil/molten globule/R-helices or β-sheets,26 as has been shown in ATR-FT-IR spectroscopy of ivory and similar solid proteins.9 However, aspects of primary structure of the solid proteins give rise to significant differences in the Raman spectra of the protein-based binding media; amino acid composition of these proteins31 and the degree to which aromatic amino acids contribute to the overall spectrum are particularly important. For example, collagen proteins which do not contain tryptophan have no peaks ascribed to this amino acid (at approximately 760 cm-1) as found in casein, egg white, and egg yolk (in milk the peak is likely hidden). However, other aromatic amino acids phenylalanine and tyrosine, present in all the proteins, are associated with vibrations at 850/880,35,36 1003, 1030, 1210, and 1605 cm-1.28 The peak at 1003 cm-1 and buried aromatic amino acid peaks at 1605 cm-1 are especially pronounced (35) Overman, S. A.; Thomas, G. J., Jr. J. Raman Spectrosc. 1998, 29, 23-29. (36) Overman, S. A.; Aubrey, K. L.; Vispo, N. S.; Cesareni, G.; Thomas, G. J., Jr. Biochemistry 1994, 33, 1037-1042.
in egg white and casein, for which phenylalanine is present in higher concentrations than in collagen-based glues31 (a total of approximately 12% of tyrosine plus phenylalanine is detected in egg and milk, compared to approximately 4% total in animal glue). Further, the presence of phosphoproteins in egg white, casein, and milk may give rise to the peak observed at 954 cm-1. It is also noteworthy that casein, which has no sulfur bridges, has no peak attributed to C-S stretching. Spectra can be discriminated by a comparison of specific peaks. A consideration of the ratio of peak intensity of phenylalanine at 1003 cm-1 (present in all proteins) relative to the amide-I stretch, for example, allows the discrimination of milk and egg white (ratio 1.7 and 1.6, respectively) from the collagen proteins (all ratios approximately 1.3); egg yolk has a ratio of 1.2 and casein 0.9, the latter due to the presence of the nonzero baseline which effectively augments the intensity of the amide-I band. In the C-H stretching region, vibrations from aliphatic C-H can be identified, as seen in Figure 3. The general shape of the C-H stretching region in six of the proteins studied is similar, with the exception of egg yolk (containing fatty Analytical Chemistry, Vol. 79, No. 16, August 15, 2007
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Figure 2. Details of spectra from Figure 1 in the region between 1800 and 500 cm-1; excitation, 785 nm; protein samples as in Figure 1. Peaks have been labeled with numbers which are given in Table 1.
acid esters) and skimmed milk (containing lactose); differences may be ascribed to nonproteinaceous components which give rise to additional peaks visible in the C-H stretching region. Casein, egg white, and the collagen proteins contain C-H vibrations which are of comparable Raman shift, but the spectral shape and background of spectra of each protein are slightly different. Weak features associated with aromatic 6148 Analytical Chemistry, Vol. 79, No. 16, August 15, 2007
C-H stretching of aromatic amino acids are apparent in egg white, casein, and milk but cannot be detected in collagen-based films due to the lower signal-to-noise ratio, as well as the lower overall concentration of aromatic amino acids in these proteins. A consideration of the C-H stretching region is useful for a number of reasons; not only are there differences in the spectra
Figure 3. Raman spectra in the region of 3200-2700 cm-1 associated with the C-H stretching of proteins; samples as in Figure 1. Significant differences are observed between different spectra, especially on the basis of shape rather than in peak position, with the exception of egg yolk (a), which has significant saturated hydrocarbons associated with fatty acid esters, giving rise to different vibrations. Peaks have been labeled with numbers given in Table 1 in parentheses.
observed among the various samples, but further, fewer interferences from other materials (especially inorganic pigments) are found. Further, the interpretation of spectra is more straightforward in this range than that of the very complex and crowded fingerprint region, hence allowing the discrimination between
complex macromolecular samples shown here. Multivariate analysis of 80 spectra in the C-H stretching range was undertaken in an effort to discriminate between the different sources of binding media studied and to determine whether or not the spectra are statistically dissimilar. Two principal compoAnalytical Chemistry, Vol. 79, No. 16, August 15, 2007
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Figure 4. Loading plots of the principal components PC1 and PC2 which account for 69% and 21%, respectively, of the total variance in the Raman spectra of the eight proteinaceous binding media studied.
nents were calculated in order to explain the variance in the data and accounted for a total of 90% of the cumulative variance in all of the samples. A loading plot of the two components is shown in Figure 4 followed by a score plot of the two principal components (Figure 5) which can be used to group proteins into six different clusters. The loading plot indicates the specific contribution of each Raman shift in the calculation of the total variance and certain spectral features can be appreciated; contributions are greatest in the region between 3100 and 2800 cm-1. In both PC1 and PC2 of the spectra considered, Raman peaks and the nonlinear background contribute to the total variance; in PC1, two peaks at 2990 and 2950 cm-1 are evident, and in PC2, a larger contribution from the aromatic amino acids at 3060 cm-1 can be observed. An inspection of the score plot shown in Figure 5 indicates that the C-H stretching region of the group of samples selected for this study is sufficiently different for each sample to allow separation of the samples on the basis of their Raman spectra featuring the C-H stretching resonances. Variations in signal-tonoise, instrumental fluctuations, and the position of analysis on
the film of the same sample are less significant than are the differences between the spectra of any two different samples. Importantly, the samples can be rapidly and easily clustered into groups; dairy proteins are found only in the bottom left quadrant of the score plot, whereas any protein found in the other areas of the plot corresponds to a collagen-based material. Although this division could also be ascertained from small differences in the fingerprint region, the clustering of the collagen proteins indicates that there is a large variation between the 40 spectra collected, but a general trend within the protein class is also observable. Specifically, the proteins extracted with water (fish glue and to a lesser extent parchment glue) without alkaline or acid treatment followed by extensive heat processing (as found with ox bone and rabbit skin glues) are separated from the other glues on the basis of both components; thus, within the cluster of collagen-based materials, fish glue can be differentiated from the other glues and appears in the top left quadrant, whereas rabbit skin glue and ox bone glue cannot be distinguished from one another. In contrast to collagen, differences between the spectra of the dairy proteins are significant and allow an immediate separation of spectra into well-defined clusters. Egg yolk and egg white, which contain similar proteins, differ due to the presence of fatty acid esters, and egg yolk is well separated from the other materials. Milk, which contains casein, is distinctly different from other samples due to the presence of other proteins and sugars within milk, which likely contribute to the largest overall variance between spectra; relatively greater variance can be ascribed to the heterogeneity of the milk protein film on the micrometer scale as well as the chemical complexity of the milk as compared to casein, similarly evident in the spectra in Figures 1 and 2. Importantly, multivariate analysis here demonstrates that Raman spectra of the C-H stretching region can indeed be used for the discrimination not only of protein class but also of the origin of the material. In an effort to test the applicability of the analytical procedure described above for the analysis of samples more similar to the mixtures of inorganic pigments with binding media found in paintings, samples containing egg white mixed with the pigment yellow ochre were considered. Raman spectra obtained (Figure 6) exhibit a strong band from the yellow ochre pigment at 387
Figure 5. Score plot of the two principal components, PC1 and PC2, of total variance between 3200 and 2800 cm-1 can be used to separate protein-based media into different clusters. Circles are ascribed to the dairy proteins from casein (red), egg white (black), egg yolk (blue), and milk (green) and squares to collagen proteins from fish glue (blue), parchment glue (red), rabbit skin glue (green), and ox bone glue (black). 6150 Analytical Chemistry, Vol. 79, No. 16, August 15, 2007
Figure 6. Raman spectrum of yellow ochre mixed with egg white. Detail of the C-H stretching region considered for multivariate analysis (inset). Peaks associated with the pigment are found at 246, 301, 387, 417 (shoulder), 482, 550, 1003 cm-1 and are marked with an asterisk (/); excitation, 785 nm.
cm-1 which is characteristic of the mineral goethite,12 and this and other pigment peaks dominate the spectrum. Small peaks related to the presence of proteins can be appreciated in the fingerprint region, but the band from C-H stretching is still observed from 3200 to 2700 cm-1 and is free from the larger spectral interferences due to the pigment which are observed in the fingerprint region. Multivariate analysis was carried out on the database of reference materials and interpolated spectra between 3200 and 2700 cm-1 from spectra recorded for the yellow ochre and egg white samples in the full spectral range. Principal component scores (PC1, PC2) of (-0.041, -0.105) and (-0.045, -0.010) fall well within the cluster determined for egg white shown in Figure 5. This demonstrates that, at least for the samples considered, it is possible to correctly identify the protein used on the basis of the C-H stretching, which was extrapolated from a well-resolved spectrum recorded of the inorganic pigment mixed with protein. CONCLUSIONS The combination of Raman spectroscopy and multivariate analysis of spectra obtained from the analysis of protein-based
films demonstrates the potential of the technique and provides a novel and additional noninvasive and nondestructive method for the identification of and discrimination among protein-based binding media used in paintings. Model films of proteinaceous materials (egg yolk, egg white, casein, milk, isinglass, parchment glue, ox bone glue, and rabbit skin glue) have been analyzed, highlighting the applicability of Raman spectroscopy even to problematic samples commonly associated with fluorescence and weak Raman scattering. Assignment of spectra revealed significant differences between peaks of different materials, further highlighted by PCA of the C-H stretching regions. Further, model paint samples containing egg white and yellow ochre were analyzed, and the protein was successfully identified using the multivariate analytical approach. It is recognized that the presence of minor quantities of fluorophores might compromise the detection and analysis of proteins using Raman, but this can be considerably minimized by selecting near-IR excitation sources. Future work should focus on the analysis of real samples to determine the actual capabilities of the proposed method and to assess the role of interferences such as fluorescence or pigment resonances. In addition, the influence of aging on protein-based materials, both with and without pigment, and the degree to which aging reactions may affect Raman spectra still require investigation. In conclusion, within the context of analysis of protein-based binding media, the use of Raman is particularly advantageous as samples are not destroyed during analysis, thus allowing further analysis of the same sample if required using alternative techniques; further, the high spatial resolution of micro-Raman spectroscopy effectively allows the qualitative analysis of submicrogram quantities of materials which is particularly powerful for the analysis of protein-based binding media. Finally, multivariate analytical methods can be powerful tools to discriminate and identify the protein media on the basis of their Raman spectra. ACKNOWLEDGMENT A. Nevin’s research was supported by the European Union Sixth Framework Programme Marie Curie Early Stage Training Fellowship as part of the ATHENA Project (MEST-CT-2004504067).
Received for review February 23, 2007. Accepted May 22, 2007. AC070373J
Analytical Chemistry, Vol. 79, No. 16, August 15, 2007
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