Combining SERS and microspectrofluorimetry with historically accurate reconstructions for the characterization of lac dye paints in medieval manuscript illuminations

8/20/2019 Combining SERS and microspectrofluorimetry with historically accurate reconstructions for the characterization of l…

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Combining SERS and microspectrofluorimetry

with historically accurate reconstructions for thecharacterization of lac dye paints in medievalmanuscript illuminations†

Rita Castro,a Federica Pozzi,b,c Marco Leonab** and Maria João Meloa*

In the present study, a number of dark red microsamples from nine illuminated manuscripts of three important medieval Portuguesemonasteries (St. Mamede of Lorvão, Holy Cross of Coimbra and St. Mary of Alcobaça) were analyzed using a multi-analytical approachto identify the dyes, fillers, binders andother additional paint components. Historically accurate reconstructions of lacdyepaints wereprepared according to recipes from medieval treatises and characterized as reference materials. Its purpose was to create better

means to study this complex dye, by using the materials and techniques as closeas possible to the medieval ones and ultimately builda solid database to support the interpretation of the results obtained from the spectroscopic techniques. Surface-enhanced Ramanspectroscopy (SERS) and microspectrofluorimetry were used here for the first time as complementary techniques to characterizelac dye paints, whereas Raman microscopy and micro-Fourier transform infrared spectroscopy were employed to detect bindersand fillers. While SERS was able to offer a conclusive molecular fingerprint of lac dye, microspectrofluorimetry provided usefulinformation on the global formulation of the red paints. Copyright © 2014 John Wiley & Sons, Ltd.

Additional supporting information may be found in the online version of this article at the publisher ’ s web site

Keywords: surface-enhanced Raman spectroscopy; microspectrofluorimetry; lac dye; illuminated manuscripts; Romanesque

Introduction

Lac is part of a resinous cocoon secreted by insects on twigs of branches of host trees. The dark red resinous raw material iscommonly called sticklac.[1,2] Lac dye, the coloring red substance,represents only 10% of the entire resin matter, and its main compo-nents are laccaic acids A and B; laccaic acids C, D and E are also foundin minor quantities[3,4] Fig. 1. In Fig. S1 (Supporting Information), thepossible acid–base forms of laccaic acid A are shown, as well as twotypes of chelating sites in themolecule. When refined, the resin givesthe well-known shellac, which is a complex mixture of monoestersand polyesters of hydroxyl aliphatic and sesquiterpenoid acids.[5]

Erythrolaccin, also shown in Fig. 1, contributes to the yellowishorange hue that characterizes the resin.[4–7]

Lac dye was mainly produced in India, Indochina and south of

China and was already known at around 1500 BC when the ancientHindu holy text Atharva Veda was written.[8,9] During the 12thcentury, lac was being imported to the western Mediterraneanthrough trade routes created by the Arabs and Jews.[10] Some of itsearliest Occidental documental descriptions are linked to Portugal,such as the one from Garcia de Orta, published in Goa in 1563,[11] orthe itinerary descriptions that Jan Huygen van Linschoten wrote afteran expedition to Goa ordered by the King of Portugal, in 1596. [12]

In the field of conservation science, lac dye has been mostly iden-tified in historical textiles.[13] Oneexception is thework conducted atthe National Gallery (London), where several notable occurrences of lac dye in paintings were reported.[4] The authors have developedan extraction method that enables the detection of the lac chromo-phores by high-performance liquid chromatography with a diode-

array detector (HPLC-DAD).[14,15] Kirby also proposes that, in addi-tion to the laccaic acids, the identification of erythrolaccin in a lacpaint may be considered as a marker for the possible presence of shellac.[4] Over the years, the characterization of lake pigmentsand dyes in works of art has greatly benefited from the advent of new advanced techniques, such as microspectrofluorimetry andsurface-enhanced Raman spectroscopy (SERS),[16–20] as well asfromthe development of improved sample pretreatments, such as non-extractive gas–solid hydrolysis procedures or mild extractionmethods.[21–23] Recent articles have shown that SERS can be used

* Correspondence to: Maria João Melo, REQUIMTE-CQFB and Department of Conservation and Restoration, Faculdade de Ciências e Tecnologia, UniversidadeNova de Lisboa, Campus da Caparica, 2829-516 Caparica, Portugal.E-mail: [email protected]

** Correspondence to: Marco Leona, Department of Scientific Research, TheMetropolitan Museum of Art, 1000 Fifth Avenue, New York, NY 10028, USA.E-mail: [email protected]

† This article is part of the special issue of the Journal of Raman Spectroscopy entitled “ Raman in Art and Archaeology 2013” edited by Polonca Ropret and JuanManuel Madariaga.

a REQUIMTE-CQFB and Department of Conservation and Restoration, Faculdade deCiências e Tecnologia, Universidade Nova de Lisboa, Campus da Caparica, 2829-516,Caparica, Portugal

b Department of Scientific Research, The Metropolitan Museum of Art, 1000 Fifth Avenue, New York, NY, 10028, USA

c Department of Conservation Science,Art Institute of Chicago, 111South Michigan Avenue, Chicago, IL, 60603, USA

J. Raman Spectrosc. 2014 , 45, 1172–1179 Copyright © 2014 John Wiley & Sons, Ltd.

Research article

Received: 30 December 2013 Revised: 12 September 2014 Accepted: 5 October 2014 Published online in Wiley Online Library: 18 November 2014

(wileyonlinelibrary.com) DOI 10.1002/jrs.4608


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successfully to unambiguously identify the main chromophore of lac dye, laccaic acid A.[18–22,24] On the other hand,microspectrofluorimetry has taken groundwork steps in the charac-terization of lac dye paints and, in particular, in providinginformation on the paint recipe used.[16] For the study of lac dyepaints, SERS and microspectrofluorimetry may be described ascomplementary techniques, considering that SERS provides aconclusive molecular fingerprint for the main chromophore,whereas microspectrofluorimetry offers valuable information onthe global paint formulation (i.e. the chromophore’s environment).In addition, the high sensitivity of microspectrofluorimetry enablesus to use the technique in situ with micrometer-level spatialresolution, unlike SERS on silver colloids that generally requiresmicrosampling. Results from both techniques need to besupported by comparison with a comprehensive spectral database,in which data from selected historical reproductions must be in-cluded. The study of documentary sources and pigment recipes isalso indispensable to properly characterize the formulationof these

paints, and so far, few researchers have exploited it.[4,14–16,25–27]

Lac dye recipes used to prepare the historically accuratereconstructions were drawn from various sources dating back tothe period between the 11th and 15th century.[28–32] The recipeswere taken from Ibn Badis manuscript, an Arabic treatise on chem-ical technology for bookmaking dated to c. 1025; Mappae Clavicula,a well known documentary source of recipes that gathers severaldocuments from the 8th to 12th centuries; O libro de komo se fazenas kores (LKFK), a Portuguese treatise from the 15th century; theBolognese manuscript from the 15th century; and finally, Strasbourg

manuscript, also from the 15th century. These treatises describetwo main preparation methods, where lac dye is either complexedwith a metal ion—typically Al3+ in the form of alum—forming alake pigment or used as a free colorant.

A first indication that lac dye may have been used for the dark red/carmine hues in Portuguese medieval manuscripts was givenin a previous work by some of the authors, based on the results of micro-Fourier transform infrared spectroscopy (μ-FTIR) analysis of aminute sample from Lorvão monastery,[25] Fig. S2 (Supporting Infor-mation). We would like to note that throughout the text, the wordcarmine will be always used as a hue, that is, an attribute for colorand not as indication for carminic acid. The presence of an organicchromophore in Holy Cross manuscripts was further confirmedwithin a MoLab mission.[33] In the present work, we perform a sys-tematic characterization of lac dye mock-up paints prepared accord-ing to medieval recipes and compare the results with data from dark reds and carmine colors found in manuscript illuminations from thefollowing Portuguese monasteries: Lorvão, Holy Cross and Alcobaça,Fig. 2.[16,25–27,33–35] During the Romanesque period (11th–13th cen-turies), these monasteries had an exceptional cultural productivity,and the paints used for the illuminations were produced in their

scriptoria, with the best colorantsavailable.[25]

Because of thesamplesize restrictions and the difficulty in achieving an efficient extractionof the laccaic acids as a result of the resin crosslinking—even usingthe extraction method developed at the National Gallery—it wasnot possible to perform HPLC-DAD analysis to characterize thedye. For this reason, microspectrofluorimetry and SERS weretested in this work as alternative methods to unambiguouslyidentify the carmine colors typical of medieval Portugueseilluminations. In this study, SERS was used for the first time inthe analysis of lac dye reds from medieval illuminations; the dataso obtained were then compared with microspectrofluorimetryresults, thus enabling to validate the latter technique as a robustanalytical tool for the detection of lac dye in situ. Raman and FTIRspectroscopies were also employed as complementarytechniques to identify binders and fillers.[35–37]

Experimental

Historically accurate reconstructions

All reconstructions were carefully prepared with distilled water, re-agent grade chemicals and sticklac from Kremer. Each recipe wasrepeated several times. The lac paints were applied on parchment

Figure 1. Chemical structures of laccaic acid A, laccaic acids B, C and E (B,R = C H2CH2OH; C, R=CH2CHNH2COOH; E, R = CH2CH2NH2), laccaic acid Dand erythrolaccin.

Figure 2. Examples of dark reds/carmine from Lorvão 5, f.6; Santa Cruz 20, f.86; and Alcobaça 419, f.98. Manuscripts at ANTT (Lisbon), BPMP (Porto) and BNP(Lisbon), respectively.

Combining SERS and microspectrofluorimetry with reconstructions

J. Raman Spectrosc. 2014 , 45, 1172–1179 Copyright © 2014 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jrs


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(acquired from the Museé du Parchemin, Rouillon), filter paper and/orglass slides withparchment glue and/or egg white as binders—whichwere the most likely used in these collections.[25,35] Parchment gluewas prepared as described in LKFK , chapter 40;[30] egg white wasmade based on the De Clarea treatise.[38]

Lac reference samples were prepared following documentedsources dating from the 11th to the 15th centuries, [28–32] adaptedto laboratory conditions. Two types of recipes will be distinguishedfor the purpose of this work, that is, free lac dyes (A) and alum–laccomplexes (B):

(A) In the Ibn Badis recipe from chapter six,[28] 5 g of ground lacwas extracted in a 50 ml solution of 0.1 g of sodium carbon-ate and borax (pH≈10), then heated, filtered and heatedagain until the ink gained thick consistency; LKFK recipe,from chapter 13,[30] uses 150ml of urine filtered in a bed of quicklime and ashes, in a proportion of 2:1 (pH ≈ 9–10);10 g of ground lac is added into the solution during heatingand then filtered.

(B) In the Mappae Clavicula,[29] 10 g of sticklac is extracted in100 ml of heated urine (pH≈6); then, 0.6g of alum (AlK (SO4)2·12H2O) is added, and the solution filtered; in theBolognese 129 recipe,[31] 150ml of one-week stale urine isheated; then, ashes are added (pH≈10). Five grams of ground lac is mixed in the solution and filtered again before0.6 g of alumis added; Bolognese 130[31] uses 60ml of 20-daystale urine (pH≈ 6–7), 10 g of finely ground lac and 2 g of alum in the same order but in different quantities (note: thisrecipe also uses brazilwood, but we decided not to include itin this study, as it would make spectral interpretation morechallenging at this stage); Strasbourg recipe named ‘BrightParis red’[32] starts by leaving 5 g of ground lac in a 50 mlof lye of ashes (pH≈11) overnight. The next day, the solutionis heated and 2g of alum is added to the mix until thelake precipitate is obtained. The solution is then filtered

and centrifuged.

In addition, we also used as reference materials for the analyticalapproach a set of reconstructions previously made in the framework of another study.[39] These were mainly prepared by extracting 3.6gor 7.2 g of ground sticklac with 30 or 60 ml of different acidic andbasic solutions, respectively. These reconstructions, not based onmedieval documentary sources, were applied on parchment withparchment glue. For the purpose of this work, these will be referredas ‘lac 1’, ‘lac 2’ and so on, as alsopart of type A free lacs, and ‘lac dyex0 precipitated’, as type B complexed lacs.

For further information on the color of the reconstructions,consult Table S1 (Supporting Information).

Other reference samples

SERS

Laccaic acids A, B, C and E were collected after HPLC-DAD separa-tion of a 103mol/l H2O : MeOH 70:30 lac dye (Fluka) solution. Theinstrumentation and solvent gradient were identical to thosedescribed in the preceding texts, although perchloric acid wasreplaced by a 10% aqueous solutionof formic acid 99.9% (v / v ). Eachlaccaic acid was collected separately into a round-bottom flask according to the retention time (several runs were performed tocollect a sufficient amount of each compound). The so obtainedsolutions were rotavapped to remove the methanol and thenlyophilized. HPLC analysis of the samples was subsequently

performed to ascertain the purity of the compounds collected. Afterthis, the individual laccaic acids were analyzed by SERS using aLabram 300 Jobin Yvon spectrometer ( λexc=633nm).

Microspectrofluorimetry

Al3+–lac complex applied on filter paper (500 μl of 103 mol/l lacdye solution in H2O : MeOH 70:30 complexed with 20 μl of 1mol/l

KAl(SO4)2·12H2O, at pH = 3.8).UV-Vis

Lac dye solutions in H2O : MeOH 70:30 prepared to a concentrationof 105 mol/l.

Historical samples

Twelve microscopic dark red samples from nine Portuguese illumi-nated manuscripts dating to the 12th and 13th centuries wereanalyzed. These manuscripts were produced in St. Mamede of Lorvão, St. Mary of Alcobaça and Holy Cross of Coimbra monaster-ies (São Mamede do Lorvão, Santa Cruz de Coimbra, Santa Maria deAlcobaça) and are currently preserved in the Arquivo Nacional da

Torre do Tombo (ANTT), Biblioteca Nacional de Portugal (BNP)and Biblioteca Pública Municipal do Porto (BPMP), respectively.Microsamples (weight less than 0.1 μg) were available from thefollowing manuscripts: Lorvão 5, ff.6 and 73v (1183), Alcobaça238, f.206v (13th c.), Alcobaça 249, f.109v (13th c.), Alcobaça 412,f.10v (13th c.), Alcobaça 419, f.98 (13th c.), Alcobaça 421, f.202(13th c.), Alcobaça 446, f.96v (13th c.), Santa Cruz 20, ff. 86 and191 (13th c.) and Santa Cruz 21, ff.2 and 19 (13th c.), Figs 2 and S3(Supporting Information).

Microsampling

Microsampling of the manuscripts was performed with a micro-

chisel from Ted Pella microtools under a Leica KL 1500 LCD micro-scope, equipped with a 12× objective and a Leica Digilux digitalcamera, with external illumination via optical fibers. As for themicrosamples taken from the paint reconstructions, a tungstenneedle was used. Microsamples were typically of 20–50μm indiameter and weight


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FTIR

Infrared spectra were acquired with a Nicolet Nexus spectropho-tometer coupled to a Continuμm microscope (15× objective) witha MCT-A detector cooled by liquid nitrogen. Spectra were obtainedin transmission mode, between 4000 and 650 cm1, with aresolution of 4 cm1 and 128 or 256 scans. Samples were prelimi-narily compressed using a Thermo diamond anvil compression cell.

Spectra are shown here as acquired, without further manipulations,except for the occasional removal of the CO2 absorption at ca.2300–2400cm1.

Microspectrofluorimetry

Fluorescence excitation and emission spectra were recorded with aJobin Yvon/Horiba SPEX Fluorog 3-2.2 spectrofluorometer. Fluores-cence spectra were corrected for the wavelength response of thesystem. For microspectrofluorimetry analyses, the latter equipmentwas hyphenated to an Olympus BX51 M confocal microscope, withspatial resolution controlled with a multiple-pinhole turret,corresponding to a minimum 2μm and maximum 60μm spot, with50× objective. Standard dichroic filters of 500 and 600nm were

used at 45° to collect the emission and excitation spectra,respectively. Emission spectra were acquired exciting at 490 nm,and excitation spectra were recorded collecting the signal at610nm. Both types of spectra were acquired on a 30μm spot(pinhole 8) and the following slits set: emission slits = 3/3/3 mmand excitation slits = 5/3/0.8mm. The optimization of the signalwas performed for all pinhole apertures through mirror alignmentin the optic pathway of the microscope, following the manufac-turer’s instructions. Spectra were collected after focusing on thesample (eye view) followed by signal intensity optimization(detector reading). Emission and excitation spectra were acquiredon the same spot whenever possible. The paint reconstructionswere mainly analyzed in situ, while the historical samples wereanalyzed in microsamples. Five spots per sample were measured

to ensure reproducibility of the results.

UV-Vis

UV-Visible absorption spectra of the paint reconstructions andreferences in a 1-cm cuvette holder were recorded with a Cary100 Bio spectrophotometer.

HPLC-DAD

HPLC-DAD analyses were carried out in an analytical ThermoElectron, FinniganTM Surveyor® HPLC-DAD system with a ThermoElectron, FinniganTM Surveyor® LC pump, Autosampler and PDAdetector and using a reversed-phase RP18 analytic column

(Nucleosil C18, 250 × 4.6 mm, 300 Å—

5μm) kept at controlledtemperature (35 °C). Samples were injected into the column via aRheodyne injector with a 25 μl loop. The elution gradient used ata flow rate of 1.7ml/min consisted of A: HPLC-grade methanoland B: 0.3% (v / v ) perchloric acid in Millipore ultrapure water. Thegradient elution program was the following: 0–2 min, isocratic 7%A; 2–8 min, linear gradient to 15% A; 8–25 min, linear gradient to75% A; 25–27 min, linear gradient to 80% A; 27–29 min, linear to100% A; and 29–40 min, isocratic 100% A.[40]

Colloid synthesis, SERS methodology and sample pretreatments

Silver nanoparticles prepared by microwave-supported glucosereduction of silver sulfate with sodium citrate as capping agent

were used as SERS substrate. A detailed description of the synthesisis reported elsewhere.[18]

SERS analysis was performed after deposition of 0.8 μl of the Agcolloid and 0.1μl of 0.5 mol/l KNO3 aqueous solution onto eachmicrosample. Spectra were collected by focusing the laser beamonto the microaggregates that formed inside the dye-colloiddroplet a few seconds after the deposition of the Ag nanoparticlesand KNO3. Several spectra were acquired continuously until thedroplet dried out.

Three sorts of SERS procedures were used, according to the typeof sample:

(1) For free lac reproductions (type A recipes), SERS analysiswas performed directly on the microsamples withoutany pretreatment;

(2) For lac–alum reproductions (type B recipes), a non-extractivegas–solid hydrolysis pretreatment was used, in which themicrosamples are exposed to hydrofluoric acid (HF) vaporin a closed microchamber for 5 min. This procedure aims tohydrolyze the dye–metal complex and increase the analyteadsorption on the nanosized metal substrate, thus enhanc-ing the SERS signal.[21]

(3) For the historical samples, because it was possible to havethe dye as free lac dye or as lac lake pigment, a two-stepprocedure was followed by analyzing the sample first with-out hydrolysis and then, after rinsing it with a water droplet,upon HF treatment.[21]

Results and discussion

Considerations on the historically accurate reconstructions

Some of the key aspects of the paint reconstructions will bediscussed in this section, because of their crucial role in interpreting

the overall results obtained in this study for the historical samples.As previously mentioned, two different types of lac pigments aredescribed in the treatises: (A) free lac dyes, characterized by the ab-sence of a complexing metal ion (described in Ibn Badis and LKFK );(B) lac lake pigments, obtained by the addition of alum (describedin Bolognese, Mappae Clavicula and Strasbourg). Non-complexedlac paints tend to be more reddish and glossy (with a pH close toneutral), while lac lake pigments develop a more pinkish color(and the solutions from where they precipitate are more acidic),Table S1 (Supplementary Information).

Most of the medieval recipes contain missing information orobscure instructions, such as omitted measured quantities orheating temperatures, which makes them open to interpretation.Bolognese, Strasbourg and LKFK recipes are perhaps the ones thatneed more extensive interpretation work, because of deficientinformation (for example, Bolognese and Strasbourg lack specific information on the quantities of alum, whereas LKFK,written in Hebrew, poses linguistic challenges that may affectits translation). As for the Mappae clavicula and Ibn Badis,recipes are significantly more detailed, which makes themeasier to reproduce accurately.

FTIR and Raman results

The dark reds (carmine) and pink colors found in the manuscriptilluminations were historically applied as a single color or as a matiz ;the pink color was identified as a mixture of a dye with white lead or




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white lead and vermilion (by Raman, infrared and microfluorescenceUV-Vis and supported by XRF[25,33,34]); some darker reds in Holy Crossmanuscripts were identified as the dye admixed with vermilion,Fig. S4 (Supporting Information).[25,34] All of these paints wereapplied with a proteinaceous binder, such as parchment glue oregg white.[35] For these carmine paints, the FTIR spectra essentiallydisplay the binder’s fingerprint as well as, in most cases, the signalsas a result of the fillers, Fig. S4 (Supporting Information). In somesamples, lac dye is indirectly detected upon identification of theC–H stretching absorption bands of the shellac resin, Fig. S2(Supporting Information). Chalk or gypsum was most commonlydetected for Alcobaça samples but was also found in a fewspecimens from Holy Cross. Because of the high fluorescence inthe Raman signal, these fillers were only detected by FTIR.

SERS results

Historically accurate reconstructions and standard laccaic acids

SERS spectra for the lac–alum reconstructions could only beobtained upon HF hydrolysis. Spectra of these reference samples(Fig. 3) displayed the typical pattern of the main component of lac dye, that is, laccaic acid A, with the most intense signals at circa1578, 1464, 1368, 1326, 1287, 1227, 1188, 1098, 1052, 1010, 453 and408 cm1 (Fig. 4(d)). On the other hand, reference SERS spectrafrom laccaic acids A, B, C and E as collected from HPLC are shownin Fig. S5 (Supporting Information). The similarity between thespectra of laccaic acid A and the commercial mixture (Figs S5aand S5b) indicates that SERS is detecting primarily the formercompound, that is, the main dye chromophore. We experimentallyacquired the relative composition of lac dye chromophores (fromFluka), by HPLC-DAD: laccaic acid A = 50%, laccaic acid B = 25%,laccaic acid C = 20% and laccaic acid E = 5% (relative areas). Overall,the best spectra were obtained for lakes that were in more acidicconditions. This is in agreement with Cañamares et al.[24] who found

that the SERS intensity for lac dye increases going from alkaline to

acidic pHs. This is likely because of theincrease in the resonance Ra-man effect combined with a lower electrostatic repulsion betweenlaccaic acid and the negatively charged nanoparticle surface(capped by the citrate ions) at acidic pH. Within this group of laclakes, minor shifts in wave number and slight changes in relativeintensities were observed, which may be attributed to pH effectsor to other components present in the matrix that may interferewith the SERS signal.

Overall, a clear distinction was observed between the lac dye–Al3+

complexes analyzed after HF treatment and the free lac reconstruc-tions examined without hydrolysis. The latter displayed intensesignals at circa 1622, 1568, 1534, 1460, 1346, 1192, 1100, 1058 and

1011 cm1

, as shown in Fig. 3. As expected from the literature,[24]

Figure 3. On the left, SERS spectra of Al3+–lac complexreconstructions, at λexc= 488 nm: (a) Mappae Clavicula; (b) Strasbourg; (c) Bolognese 130; (d) Bolognese

129. On the right, SERS spectra of non-complexed lac reconstructions, at λexc=488nm: (e) Ibn Badis 2; (f) Libro de komo se fazen as kores; (g) lac dye 3reconstruction (pH≈ 3); (h) sticklac (raw material).

Figure 4. SERS spectra of the historical samples, at λexc= 488 nm: (a)Lorvão 5, f.6; (b) Santa Cruz 20, f.191; (c) Alcobaça 446, f.96v; (d) laccaicacid A at pH= 2.

R. Castro et al.

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in samples where the HF treatment was applied (acidic media) theband at 1464 cm1, assigned to C–C I ring stretching modes and toC8–OH and C5–OH bending modes, displayed the highest intensity.On the other hand, in the samples analyzed without HF (neutral tobasic media), the highest intensity was observed at 1340–50cm1.In the literature, this latter band is normally assigned to the depro-tonation of the C5–OH bending mode in the molecule, Table S2(Supporting Information).[22,24]

Historical samples

As far as the historical samples are concerned, while regular SERSproved unsuccessful as a result of interference of the citrate ionscapping the nanoparticle surface, the use of the HF pretreatmentensured certain dye identification in all cases. This approachenabled us to confirm that the dark red used in PortugueseRomanesque illuminations was based on lac dye, Fig. 4 and TableS3 (Supporting Information). The fact that SERS only worked afteracidic treatment suggested that the historical paints from themanuscripts may have been all complexed with a metal ion;however, cross-linking of the resin may also have prevented the

dye from being effectively mobilized in the paint sample withoutHF hydrolysis. Overall, the SERS spectra of the historical samplesare very similar to the lake reproductions spectra, revealing onlyslight variations in the 1580–1010 cm1 region. Interestingly,Mappae Clavicula (Fig. 3(a)) and Bolognese type B reconstruction(Fig. 3(c) and 3(d)) spectra showed an excellent resemblance withthespectrum obtained from Lorvão5, f.6 historical sample (Fig. 4(a)).

Microspectrofluorimetry results

As the need of HF pretreatment for all of the historical samplesexamined suggested the use of lac–alum pigments in themanuscript paints, in this section, special attention will be devotedto reconstructions based on recipes in which lac dye is complexedwith Al3+ ions. Before examining the paint reconstructions though,the main spectral features of raw sticklac and shellac resin will bediscussed. Figure S6 (Supporting Information) shows the emissionand excitation spectra of the two materials. In the spectra of rawsticklac, both the resin and all the lac chromophores are detected,whereas for shellac, the main contribution observed is that of theresin itself. The laccaic acid chromophores dominate the spectrafor the raw material, and the excitation spectrum is characterizedby a broad, unresolved band, with a maximum at 505 nm. Note thatin all excitation spectra presented, showing the absorption of theemitting species, for very low intensities, the band at circa 420nmis an instrumental artifact. At 490nm excitation, the emissionspectrum of sticklac mainly displays features of the laccaic acids.

The shellac resin’s excitation spectrum displays a band at 463 nm,

which compares well with the absorption spectrum published inliterature for erythrolaccin,[4] characterized by maxima at 264, 294,464 nm, of which only the latter can be detected with ourinstrumental setup (dichroic filter 600 nm).

On the other hand, the Al3+–lac dye complex solution applied tofilter paper, at pH = 3.8, displayswellresolved spectral features, witha small Stokes shift, indicating that the same species is absorbingand emitting in the excited state (Fig. 5(a)). The excitation spectraare characterized by two bands, with maxima at 526 and 562 nm,in agreement with the absorption spectra. The relative intensitiesof the two emission bands may change according to the pH, andminor shifts may occur depending on the amount of alum present.Close to neutral and basic pH, the signal is lower and the band at

526 nm displays the highest intensity. The emission spectrum, notso finely resolved, displays a maximum at 593 nm, Fig. 5(a).

When comparing the references discussed in the preceding textswith the historically accurate reconstructions (Table 1; refer to Fig.S7 (Supporting Information), for representative fluorescencespectra), the Bolognese 130 recipe is very similar to the Al3+–laccomplex solution described previously, with a slight shift in theemission spectra. In Mappae Clavicula, the main features in bothemission and excitation spectra are maintained, although in the

Figure 5. Excitation and emission spectra from (a) the Al3+–lac complex

(―) on filter paper; and its absorption spectra (….) in solution (105 mol/l);(b) Lorvão 5, f6 (―) with Mappae Clavicula recipe (---); (c) Alcobaça 412,f.10v (―) with Ibn Badis recipe (---), respectively.




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excitation spectra, the bands are not resolved and the relativeintensities inverted, being the band around 520–525nm the mostintense. The shoulder at circa 470 nm may include the contributionof erythrolaccin and can also be because of a lower ratio of alum–lac complex. For the Ibn Badis reproduction, the main band in the

excitation spectrum is shifted toward lower wavelengths and islocated at 472 nm. This may be ascribed to the presence of theerythrolaccin as well as to the completeabsence of alum. Giventhatin the literature, the detection of erythrolaccin is usually associatedwith the presence of shellac resin, we may conclude thatmicrospectrofluorimetry is indirectly sensing the presence of theresin and indicating that the latter was incorporated in the finalpigment.[4] To confirm this hypothesis, HPLC-DAD was performedon the lac reproductions. While the chromatogram of Bolognese130 reconstruction showed only the main complexed laccaic acidchromophores at 490 nm, the Ibn Badis was found to contain resinas well, Figs S8 and S9 (Supporting Information), in accordance withthe microspectrofluorimetric data.

The emission and excitation maxima for the original medieval

samples are displayed in Table 2. The emission spectra show a bandaround 589nm, which is detected consistently for most of the lacreproductions. As observed in the reconstructions, more pro-nounced shifts are observed for the excitation spectra. In addition,some of the original samples display a band at 472 nm, particularlyprominent in the Alcobaça samples, which, as discussed in thepreceding texts, is likely because of the presence of higher amountsof resin. In terms of signal intensity, more pronounced emissionsare observed in samples from Lorvão (circa 2×), whereas spectra of Alcobaça samples are characterized by lower intensities. Whencomparing these with laccaic acids, lac–Al3+, sticklac and shellacspectra, it is possible to conclude that the original samples arecomposed of laccaic acids and shellac, Figs 5 and S6 (Supporting

Information). Compared with the reconstructions, the most signifi-cant similarities were detected between the historical samples andMappae Clavicula—also in accordance with the SERS results—andIbn Badis mock-ups (Fig. 5(b) and 5(c)). The color of these two recon-structions is also very similar to that observed in the manuscripts.

Conclusions

In this work, for the first time, lac dye was unequivocally identifiedin medieval illuminations. SERS proved to be an advantageoustechnique for the identification of dyes in illuminated manuscripts,because of the minute sample size requiredfor analysis (10–20μm).By using the unambiguous molecular fingerprint of the dye

provided by SERS, we were able to assess the microspectro-fluorimetry results and validate this technique as a robust analyticaltool for the detection of lac dye in situ. Work is in progress to furtherexplore the applications of microspectrofluorimetry.

The availability of historicallyaccurate reconstructionswas essen-

tial for this study, as it enabled to assemble a consistent spectraldatabase of mock-ups prepared according to historical recipes, tobe used for comparison in the analysis of samples from actual me-dieval illuminations. Additionally, comparison with paint recon-structions prepared in the lab shed new light on the fact that thecolor shades observed in the medieval illuminations (pink, redand violet, to brownish hues) may be the result of the processingof the colorant as opposed to being as a result of degradation.

While SERS provides unequivocal identification of laccaic acid A(the main component of lac dye), microspectrofluorimetry de-scribes the chromophore in its environment. In this sense, thesetwo techniques, combined in this study for the first time, can beseen as complementary. The microspectrofluorimetric data ob-tained so far suggest that it is also possible to detect the resin in

a lac sample indirectly by using erythrolaccin as a marker; thiswas further confirmed by HPLC-DAD.

As for future research, we intend to explore the use of mixtures inlac paints, such as fillers (gypsum, chalk), vermilion, lead white andseveral dyes, in order to establish correlationswith the historical sam-ples. To elaborate the spectral information obtained by SERS andmicrofluorimetry and ascertain whether it may carry details on theprocess used to prepare the pigment or other hidden patterns andfeatures, a multivariate analysis approach will be followed.

In conclusion, our research sheds new light on the use of thishistorical dye. Its systematic application in Portuguese medievalmanuscripts to obtain dark red colors shows the importance of lac dye throughout the 12th and 13th centuries (much earlier than

the arrival to India of the Portuguese explorer Vasco da Gama, in1498). There is no evidence, to date, that lac dye was being usedby medieval monasteries other than those cited in this work. Thusfar, it has only been identified by SERS in a French polychromewood sculpture[18] and in a Spanish crucifix,[22] both dating to1150–1200. These three occurrences, in the Iberian Peninsula andProvence, confirm the existence of an Arabic and Jewish tradenetwork in that region during the Romanesque period.

Acknowledgements

This work has been financially supported by Portuguese fundsthrough FCT —Fundação para a Ciência e a Tecnologia under theprojects PTDC/QUI-QUI/099388/2008 and PTDC/EAT-EAT/104930/

Table 2. Fluorescence emission and excitation maxima for representative manuscripts

Lv 5 ALC 238 ALC 249 ALC 412 ALC 419 ALC 421 ALC 446 SC 20 SC 21

λ em/nm 587–89 563 589 587 587 593 593 587–89 589–91

λ exc/nm 523–26 522 554 474 556 470 476 553 520

Table 1. Fluorescence emission and excitation maxima for the main paint reconstructions

Ibn Badis Mappae Clavicula LKFK Bolognese Strasbourg

λ em/nm 589 590 No signal 589 587

λ exc/nm 472 520 No signal 526, 562 526, 562

R. Castro et al.

wileyonlinelibrary.com/journal/jrs Copyright © 2014 John Wiley & Sons, Ltd. J. Raman Spectrosc. 2014 , 45, 1172–1179


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2008. We thank REQUIMTE for supporting the project PEst-C/EQB/ LA0006/2013 and Rita Castro FCT-MEC with a PhD grant (SFRH/ BD/76789/2011). The authors would also like to thank the staff and directory board of Arquivo Nacional da Torre do Tombo(ANTT),Biblioteca Nacional de Portugal (BNP) and Biblioteca Pública Munic-ipal do Porto (BPMP) for their generous support and collaboration.

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Combining SERS and microspectrofluorimetry with historically accurate reconstructions for the characterization of lac dye paints in medieval manuscript illuminations