Talanta 80 (2009) 286–293

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Excitation emission and time-resolved fluorescence spectroscopy of selectedvarnishes used in historical musical instruments

Austin Nevin a,∗, Jean-Philippe Echard b,c, Mathieu Thoury d, Daniela Comelli a,Gianluca Valentini a, Rinaldo Cubeddu a

a ULTRAS-CNR-INFM and IFN-CNR, Politecnico di Milano, Dipartimento di Fisica, Piazza Leonardo da Vinci 32, I-20133 Milan, Italyb Laboratoire de Recherche et de Restauration, Musée de la Musique, Cité de la Musique, 221 avenue Jean Jaurès, 75019 Paris, Francec Centre de Recherche sur la Conservation des Collections, CNRS UMR 7188, Muséum National d’Histoire Naturelle, 36, rue Geoffroy Saint-Hilaire, 75005 Paris, Franced Scientific Research Department, National Gallery of Art, 6th & Constitution, Washington, DC, 20565, USA

a r t i c l e i n f o

Article history:Received 26 March 2009Received in revised form 12 June 2009Accepted 29 June 2009Available online 4 July 2009

Keywords:VarnishTerpenoidFluorescence spectroscopy

a b s t r a c t

The analysis of various varnishes from different origins, which are commonly found on historical musicalinstruments was carried out for the first time with both fluorescence excitation emission spectroscopyand laser-induced time-resolved fluorescence spectroscopy. Samples studied include varnishes preparedusing shellac, and selected diterpenoid and triterpenoid resins from plants, and mixtures of these materi-als. Fluorescence excitation emission spectra have been collected from films of naturally aged varnishes.In parallel, time-resolved fluorescence spectroscopy of varnishes provides means for discriminatingbetween short- (less than 2.0 ns) and long-lived (greater than 7.5 ns) fluorescence emissions in eachof these complex materials. Results suggest that complementary use of the two non destructive tech-niques allows a better understanding of the main fluorophores responsible for the emission in shellac,and further provides means for distinguishing the main classes of other varnishes based on differences in

Fluorescence lifetime

Musical instrument fluorescence lifetime behaviour. Spectrofluorimetric data and time resolved spectra presented here mayform the basis for the interpretation of results from future in situ fluorescence examination and time

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. Introduction

Fluorescence spectroscopy is particularly useful for the exam-nation of works of art and historic musical instruments, as manyinding media [1–5], modern pigments [6] and colorants [7] may be

uminescent. With the advantage of not requiring any sample andeing non-contact, fluorescence spectroscopy can also be coupledith multispectral and time-resolved fluorescence imaging tech-

iques for the analysis of painted surfaces [8–10], thus increasinghe utility of fluorescence spectroscopy for the characterisation of

aterials and works of [11]. In this work, time resolved and fluores-ence excitation emission spectroscopy have been employed for therst time for the analysis of selected musical instrument varnishes.

The conservation and assessment of musical instrument var-ishes may require the characterisation of their constituent

aterials. Varnishes represent a sensitive interface between

ainted or unpainted wooden surfaces and their environment, serv-ng to protect an instrument, as well as to enhance and changehe object’s appearance. While paintings and musical instruments

∗ Corresponding author. Tel.: +39 0223996010; fax: +39 0223996126.E-mail address: austinnevin@gmail.com (A. Nevin).

039-9140/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.talanta.2009.06.063

of varnished musical instruments.© 2009 Elsevier B.V. All rights reserved.

are significantly different in terms of their use, history and tech-nique, parallels between the materials used for the constructionand varnishing of historical string instruments and polychromy areapparent [5]. Paintings are often coated with multiple and thin lay-ers of natural resins (from a variety of animal and plant sources)dispersed in a solvent as a varnish or surface coating, as describedin historical treatises on painting and gilding [12], and varnishesare often applied to paintings during conservation or restoration.Recent analysis of Leonardo’s Mona Lisa has suggested the presenceof multiple layers of oxidised and yellowed varnish, which signifi-cantly alter the appearance of the masterpiece, but which have beenretained for their historical value [13]. On the other hand, an integralpart of the construction of stringed instruments (including lutesand violins, among others) is the varnishing and finishing of thewooden surface, which contributes to the appearance (colour andgloss) and to the protection of an instrument, and, hypothetically,to the quality of sound produced [14].

The presence of different varnishes and other conservation

materials on musical instruments is easily appreciable due to thevariation in fluorescence emissions from the surface of instrumentswhich may increase with age [1]. In fact, due to their compo-sition and degradation, many varnishes are naturally fluorescentor develop fluorescence upon ageing (which may also be accom-

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anied by yellowing). The visual assessment of the fluorescencerom musical instruments (as well as paintings) with UV excita-ion using a Wood lamp (emitting a broad band around 365 nm) isidespread, providing immediate indications to conservators and

urators regarding the potential presence of retouching, repaintingnd surface heterogeneity. For example, it is common to inspecthe abrasion or wear on the back of some stringed instruments,here varnish may be consumed due to cumulative use. Indeed,

ome violin experts claim that they can recognise specific “fluoresc-ng colours” on varnishes of famous violins from Cremona, whichnclude those by Amati and Stradivari.

Although UV-excitation is commonly used in museums for thexamination of musical instruments, the spectrofluorimetric anal-sis of varnishes found on the surfaces of stringed instrumentsnd paintings is relatively limited: laser induced fluorescence (LIF)f varnishes has highlighted significant differences in emissionsf common varnishes [2,15] and recently spectrofluorimetry haseen proposed for the differentiation between sandarac, mastic andammar [4]. Because of the complex chemistry and large numberf sensitive molecules present in most varnishes it has not beenossible to attribute or fully interpret fluorescence spectra.

In most cases, the determination of the chemical compositionf varnishes is carried out using sensitive and highly specific chro-atographic/spectrometric analysis [16–20], providing indications

egarding original composition, mixtures of materials and degra-ation mechanisms associated with ageing and light exposure;

ndeed, results from analysis form the basis for the differentiation ofany organic materials, including varnishes, and also provide the

ramework for the rationalisation of results from other complemen-ary spectroscopic techniques, including fluorescence spectroscopyn this work. Alternative and complementary non-destructive tech-iques, which can provide information about the surface of musical

nstruments, are particularly advantageous; sampling is not limitedy access to fragments, and when it is possible to integrate punctualnalysis with imaging, more representative indications regardinghe composition surfaces may be obtained. The specificity of moston-invasive techniques can be limited to the elemental level (forxample X-ray fluorescence spectrometry) [21] or to the detectionf broad classes of molecular materials (using Fourier-transform

nfrared spectroscopy) [22].In this work, fluorescence spectroscopy integrated with time-

esolved fluorescence analysis is investigated on model samplesf vanishes. For a more complete visualisation of the contributionrom multiple-fluorophores present in the complex systems foundn varnishes, where variations in fluorescence may be small andot related directly to the position of the maximum [4], maps ofhe fluorescence emissions as a function of excitation wavelengthave been generated. Studies of different materials from food haveemonstrated the potential of fluorescence excitation emission (EE)pectroscopy, in which a series of emission spectra that have beenollected over a range of excitation energies, allowing the discrim-nation between complex multiple protein [23] and multiple oil-ased systems [24]. In addition to EE spectra, the modelling of theanosecond decay of fluorescence can be particularly helpful for thenalysis of complex systems which contain multiple fluorophores,s has been demonstrated for binding media used in paintings [11].arious portable instruments based on LIF devices have been devel-ped and time-resolved fluorescence spectroscopy and imagingave been used to examine complex inorganic and organic paintedurfaces [9] and sculptures [25], with significant advantages for theetection of the presence of superimposed organic materials. How-

ver no time-resolved fluorescence spectra of varnishes found onaintings or musical instruments have been published.

Therefore, in this work, both fluorescence EE spectra and time-esolved fluorescence spectra of varnishes are presented for the firstime. Films of selected varnishes were prepared according to his-

0 (2009) 286–293 287

torical treatises [12,26] by a lacquer conservator at the Musée dela Musique to replicate typical thickness and surface morphologyof varnishes found on musical instruments. The aim of this work isto measure fluorescence properties of these materials and to assessthe potential of such measurements for material classification, thusproviding a useful framework for the interpretation of fluorescenceimaging of instruments.

2. Experimental

2.1. Description of samples

The materials analysed in this work were specifically chosento reflect commonly found varnishes used for historical musicalinstruments. While most varnishes typically used in violin man-ufacture [26,27] are obtained from plants, shellac is excreted bythe Kerria lacca insect and may be red-brown in colour prior topurification. Two different shellac samples have been consideredin this work: cherry shellac which has not been extensively puri-fied and which yields a slightly yellow varnish film, and platinashellac, which is bleached and colourless, having been washed andtreated prior to use. Other varnishes studied can be broadly groupedinto two categories based on their terpenic chemical composition.The triterpenoids considered are elemi and mastic. Diterpenoid var-nishes included colophony, Venice turpentine, sandarac, and copalfrom two sources, Manila (Philippines) and Congo.

For this research samples have been prepared from the restora-tion materials stored at the laboratory of the Musée de la Musiqueand purchased more than 10 years ago from local suppliers (Pro-labo, HMB and Art & Conservation, Paris). Most of these materialshave been previously analysed with GC/MS [14].

Varnish solutions have been prepared by dissolving each ofthe following resins in ethanol (1:2, w/w), and undissolvedresidual materials were decanted. Further, mixtures of equalparts of colophony:sandarac, colophony:elemi, elemi:sandarac andcolophony:elemi:sandarac were also prepared. Varnishes werepainted out in single layers on non-fluorescent quartz slides. Thechoice of a smooth non-fluorescent substrate for an assessment offluorescence of varnishes is important for two main reasons. First,only fluorescence from the varnish (and not from the substrate)is assessed. Second, coloured surfaces may selectively reflect pho-tons generated during fluorescence, which may compromise thedetection of fluorescence emissions [28].

Varnishes were prepared in 2006 and were analysed with flu-orescence devices in 2008. Therefore, while natural and artificialageing are both recognised as significant factors in the oxidation ofvarnishes [18], which may influence fluorescence emissions [1,4],for this work only naturally aged reference samples have been con-sidered in an effort to assess if fluorescence spectroscopy can allowthe differentiation between naturally aged materials.

2.2. System set-up and data analysis

2.2.1. Fluorescence excitation emission spectroscopyEE spectra of films of varnishes were acquired using a scan-

ning spectrofluorimeter with excitation provided by a Xenon arclamp (Fluorolog, Jobin-Yvon/Horiba); a front-face geometry wasemployed during collection (detection at 23◦ with respect to theexcitation slits) and the detected signal was automatically cor-rected for excitation and detection efficiency. Spectra matrices were

constructed by recording a series of synchronous fluorescence spec-tra with variable excitation between 300 and 500 nm (resolutionof 1 nm), with synchronous fluorescence offsets between 10 and250 nm in 5 nm steps, and with slits of 1 nm (excitation) and 5 nm(emission). The precision of the measurement is thus ±1 nm for the

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xcitation monochromator and ±5 nm for the emission wavelengthand hence ±6 nm for Stokes Shift). Matrices were then transformednto EE spectra using Matlab (Mathworks) and have been plot-ed as contour plots with 20 different levels of intensity from the

aximum (white) to 10% of the maximum intensity (black). Theaximum peak is defined by the excitation and emission wave-

engths at the maximum emission intensity. Peaks are reported inhe text as (�excitation/nm:�emission/nm).

.2.2. Time-resolved fluorescence spectroscopyTime-resolved fluorescence spectra were recorded using an

ptical Multichannel Analyser working in nanosecond gated mode,s described previously [9] and briefly summarised below.

UV pulsed excitation was provided by a nanosecond N2 laserith 337 nm excitation; the emitted fluorescence was collected

hrough a monochromator, with sensitivity between 400 and50 nm and a spectral resolution of 1 nm, coupled to a time-gatedultichannel analyser. The time-resolved detector is associatedith a temporal gate of 10 ns and a jitter below 0.5 ns.

The kinetics of the emission was analysed by recording a setf fluorescence spectra with increasing delays with respect tohe excitation pulse. Following data acquisition, spectral data areutomatically corrected for the detection efficiency and are pre-rocessed with a moving average smoothing filter in order to reducehe level of noise. The fluorescence decay is then analysed at eachavelength by using a procedure based on a non-linear regression

east-squares fit.When using conventional fluorimetric techniques, the pres-

nce of a variety of fluorophores in complex fluorescent materialss indicated by the dependency of the fluorescence emission onhe excitation wavelength. Moreover, the fluorescence spectra areomposed of one or several broad band emissions generated by con-ributions from numerous fluorophores. In terms of fluorescenceecay, a complex fluorescent material commonly shows a multi-xponential decay where the emission from each one of the mainmitting species or group of fluorophores can be modelled as aingle-exponential decay characterized by a specific lifetime andmission. Hence, the analysis of the decay kinetics of the emissionpectrum of a complex material can provide a way for distin-uishing amongst its main emitting fluorophores characterized byifferent lifetimes.

In this work, the analysis of the nanosecond emission decayollowing excitation at 337 nm was performed considering threeifferent models: a mono-, a bi- and a tri-exponential decay.or all the selected materials, whereas the application of the bi-xponential decay model highly improves the result of the fittingrocedure with respect to the mono-exponential model, the usef a tri-exponential decay model does not yield significant infor-ation. Therefore, in the following section, only results from the

pplication of a bi-exponential model will be discussed.Specifically, the application of a bi-exponential fitting procedure

ssumes that the optical fluorescence fluence f(t, �) which is emit-ed t seconds after the excitation pulse at the emission wavelength

can be modelled as:

(t, �) = ASHORT(�) exp( −t

�SHORT(�)

)+ ALONG(�) exp

( −t

�LONG(�)

)

(1)

here ASHORT (�) and ALONG (�) indicate the wavelength-dependentmplitudes of the short-lived and long-lived fluorescent compo-ents identified in the emission, and �SHORT (�) and �LONG (�) are

he associated wavelength-dependent lifetimes. Following a suit-ble non-linear regression least-squares fitting procedure, ASHORT�), ALONG (�), �SHORT (�) and �LONG (�) are estimated, and the meaneduced �2 value is used for assessing the reliability of the fit. Gen-rally, with this model, while the wavelength-dependent functions

0 (2009) 286–293

ASHORT (�) and ALONG (�) are associated with spectral shapes char-acterized by distinct spectral features, �SHORT (�) and �LONG (�) areassociated with relatively constant spectral behaviour. In principle,ASHORT (�) and ALONG (�) describe the emission spectrum of thetwo main emitting groups of fluorophores present in each one ofthe materials studied.

In the following section, the outcome of the bi-exponential fit-ting procedure is shown in terms of ASHORT (�) and ALONG (�), which,for an easier comparison, are always shown normalized to theirmaximum value. The mean lifetime �SHORT and �LONG associatedwith each one of the reconstructed components is further indi-cated, together with the ratio R between the total intensity of theshort-lived and long-lived component, calculated according to thefollowing formula:

R =∫

ASHORT(�) d�∫ALONG(�) d�

(2)

Hence, in each varnish studied, in addition to picosecond life-time components that cannot be resolved with the instrumentationemployed here, two main emitting groups can be well separated.

3. Results

EE spectra of the materials studied can be classified based on thenumber of evident peaks in the contour plots, the position of themaximum emission and excitation wavelengths and the generalshape of the excitation/emission contours present in the spectra.Spectra below can be divided into three basic groups: those from (1)shellac, (2) triterpenoid and (3) diterpenoid varnishes and are pre-sented in this order in the following subsections. Finally the EE andtime-resolved spectra of mixtures of selected varnishes are shown.

3.1. Shellac

Shellac is composed of a resinous fraction and of a solid colorantfraction, containing various laccaic acids. The differences betweenthe distinctly red cherry shellac and the colourless platina shel-lac are likely due to the extraction of red-coloured laccaic acidsduring the washing and bleaching process associated with the pro-duction of platina shellac. EE spectra of purified (platina) shellac,and red-coloured (cherry) shellac are seen in Fig. 1. Although ofsimilar origin, the different shellac varnishes exhibit significantlydifferent EE spectra. Specifically, while the former is associatedwith one broad emission centred at (307:487), as well as signifi-cant fluorescence at longer excitation wavelengths, the latter hasmultiple fluorescence bands: a broad fluorescence pattern cen-tred at (307:410) with the addition of intense peaks at (307:554)and (465:635) [2,29]. The last two peaks may be attributed tothe fluorescent laccaic acids. The fluorescence of shellac is clearlydependent on its degree of purification.

The analysis of the kinetics of the fluorescence emission decayconfirms the differences ascribable to the two materials and furtherprovides indications on the presence of secondary fluorescent com-pounds in each one of the two different shellacs studied. In detail,in Fig. 2 the fluorescence spectra of the short- and long-lived com-ponents of the emission from the two shellacs are shown: emissionfrom the purified platina shellac is characterised by a short-livedcomponent with a major peak at 420 nm and a further broad emis-sion centred around 600 nm, possibly ascribable to residual laccaicacids. In the long-lived component the red-emission almost dis-

appears and instead a broad emission at 440 nm with a shoulderat 520 nm is evident. Cherry shellac is characterised by an intenseshort-lived component with two peaks at 420 and 600 nm, simi-larly to those observed in the platina shellac, but in this case theemission ascribed to laccaic acids is predominant. The long-lived

A. Nevin et al. / Talanta 8

Fig. 1. EE spectra of naturally aged films of varnishes made from (a) shellac (platina),(b) shellac (cherry); intensity is given in counts and 20 contours represent 90% ofthe fluorescence of materials as a function of excitation.

Fig. 2. Fluorescence spectra of the short- and long-lived components of the emissionfrom naturally aged films of shellac following a bi-exponential fit of time-resolvedspectral data: (a) short-lived and (b) long-lived component of platina shellac; (c)short-lived and (d) long-lived component of cherry shellac. For an easier comparisonfluorescence amplitudes are shown normalized to the maximum value and valuesof �SHORT, �LONG and R are provided. Dashed vertical lines indicate the main peakwavelength of each fluorescent component.

0 (2009) 286–293 289

component of the emission of cherry shellac shows further emis-sion in the red with a peak at 630 nm, again possibly ascribable tolaccaic acids.

3.2. Triterpenoid varnishes

The EE spectra of two triterpenoid varnishes elemi and mas-tic (Fig. 3) reflect small difference in their chemical composition,which may be related to the presence of specific terpenic acids[17]. In detail, EE spectra of elemi and mastic have symmetricalfluorescence emission bands and both varnishes are associatedwith maximum emission at excitation around 310 nm. Howeverthe emissions of the two varnishes differ significantly, with Stokesshifts of 85 and 135 nm, respectively, suggesting that the materi-als are characterised by a different group of fluorophores whichmay have similar absorption spectra. The EE spectra of mastic andelemi also indicate the presence of significant visible fluorescenceat longer excitation wavelengths and the shape of the contours isrelatively similar for both materials. While specific fluorophoreshave not been identified for either varnish, these results are compa-rable with fluorescence reported by others who suggest emissionsin mastic at 440–460 nm [1,4,15]. Moreover, excitation at 365 nmof samples of mastic aged naturally for 50 years has been reportedwith emission spectra similar to those represented above [1]. It isfurther of interest that elemi and platina shellac have a common

excitation-emission maximum (as can be observed in Table 1), butEE spectra of the two varnishes are completely different.

The analysis of the kinetics of the emission at 337 nm excita-tion in shown in Fig. 4. Fluorescence emissions from elemi appear

Fig. 3. EE spectra of naturally aged films of varnishes (a) elemi (b), mastic; intensityis given in counts and 20 contours represent 90% of the fluorescence of materials asa function of excitation.

290 A. Nevin et al. / Talanta 80 (2009) 286–293

Table 1Fluorescence properties of samples examined with continuous wave excitation between 300 and 500 nm and with pulsed excitation at 337 nm.

Sample �ex max(nm)

�em max(nm)

Stokes shift(nm)

�em max(nm) ofASHORT (�)

�em max(nm) ofALONG (�)

Mean �SHORT

(ns)Mean �LONG

(ns)Mean reduced �2

value of thebi-exponential fit

Mean reduced �2

value of themono-exponential fit

Shellac (cherry) 307 410 103 430 580 2.0 8.4 0.009 0.112307 554 247 600 640465 635 170

Shellac (platina) 307 387 80 425 440 1.5 8.2 0.013 0.270600 625

Elemi 310 395 85 410 480 1.9 8.8 0.006 0.125Mastic 310 445 135 430 460 1.8 8.0 0.009 0.071Colophony 310 505 195 420 530 1.7 7.7 0.010 0.170Venice turpentine 305 465 160 410 480 1.8 9.0 0.010 0.133Sandarac 320 415 95 400 460 2.1 7.5 0.004 0.050Copal (Manila) 314 389 75 405 465 2.0 7.5 0.004 0.061Copal (Congo) 316 401 85 430 450 1.8 8.7 0.012 0.212Colophony:sandarac 313 445 132 415 485 1.9 8.2 0.007 0.079C 90E 60C 80

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haracterised by an intense short-lived component with a strongnd narrow peak at 410 nm, whereas in the long-lived componentbroader emission centred at 480 nm is evident. In turn, mastic is

haracterised by a short-lived component peaked at 430 nm with ahoulder at 410 nm and a long-lived component peaked at 460 nm,hich, compared to that of elemi, is much narrower. Nevertheless,

espite some detectable differences in their time-resolved spectra,he two materials clearly show common features related to theirimilar chemical composition. Moreover, differences between theesults of time-resolved analysis of triterpenoid-based and shellac-ased materials indicate these two classes of varnishes can beistinguished easily.

.3. Diterpenoid varnishes

The EE spectra of the diterpenoid varnishes analysed (Fig. 5)ary in terms of the position of the emission maximum, withhe exception of sandarac and copal. Moreover, in comparison

ig. 4. Fluorescence spectra of the short- and long-lived components of the emissionrom naturally aged films of varnishes following a bi-exponential fit of time-resolvedpectral data: (a) short-lived and (b) long-lived component of elemi; (c) short-livednd (d) long-lived component of mastic. For an easier comparison fluorescencemplitudes are shown normalised to the maximum value and values of �SHORT, �LONG

nd R are provided. Dashed vertical lines indicate the main peak wavelength of eachuorescent component.

1.9 8.2 0.011 0.1602.0 8.4 0.005 0.0882.0 8.6 0.006 0.071

with the emission from triterpenoid varnishes, the distribution offluorescence is significantly different; specifically, more intense flu-orescence is observed at longer excitation wavelengths, providingan immediate means of distinguishing between diterpenoids andtriterpenoids analysed.

The most distinctive and complex EE spectrum of thediterpenoid-based varnishes studied in this work is associated withcolophony, the solid residue obtained after filtration and distillationof pine turpentine. Indeed, two different groups of fluorophoreshave been assigned to the bands present: (370:480) and anotherstronger peak at around (310:505). Multiple groups of fluorophoreswhich are characterised by different bands are likely present in thesample. In contrast, the resin from larch trees, commonly known asVenice turpentine, is associated with a similar maximum emissionband but has fewer contributions to fluorescence at longer excita-tion wavelengths. This phenomenon may be related to variationin extraction and processing of the resins or to variation in thepolymerisation of the varnish films [20].

Sandarac and copals (Fig. 5(c and d)) are other diterpenoidconifer resins, and form cross-linked fraction of polycommunicacids upon oxidation, but differ in terms of the age of the collectedtree-resins prior to their use as varnishes. Sandarac is harvesteddirectly from the tree while copal is a semi-fossilised resin collectedfrom the ground and the two materials share common chemicalmarkers: agathic acid and related labdane compounds are pre-dominant in copals, and are also present in sandarac [30]. Onlysubtle differences are observed in the EE spectra of copal and san-darac which are associated with the shape of contours describingfluorescence, and spectra of the two varnishes share a commonmaximum at approximately (310–20:390–415). Small differenceswere found in the EE spectra of the two copal-based varnishes(Congo copal not shown); rather than a shift in the position of thefluorescence maximum, fluorescence emissions excited at wave-lengths above 400 nm are more apparent in Congo copal (fromCopaifera guibourthiana) which has a spectrum more similar to san-darac than to that observed in Manila copal (from Agathis dammar),likely due to the differences in the source and age between thetwo semi-fossilised copals. Published spectra of aged sandarac arecomparable to those represented in EE spectra [4].

The time-resolved analysis of the emission performed on the five

diterpenoid varnishes (Fig. 6) indicates the presence of commonfeatures for these materials, ascribable to their similar chemicalcomposition: in particular, it is possible to observe the presenceof a common short-lived and intense component peaked between400 and 420 nm, characterized by a much broader emission with

A. Nevin et al. / Talanta 80 (2009) 286–293 291

Fig. 5. EE spectra of naturally aged films of varnishes (a) colophony (b) Venice tur-pentine (c) sandarac (d) Manila copal, where intensity is given in counts and 20contours represent 90% of the fluorescence of materials as a function of excitation.

Fig. 6. Fluorescence spectra of the short- and long-lived components of the emis-sion from naturally aged films of varnishes resulting following a bi-exponentialfit of time-resolved spectral data: (a) short-lived and (b) long-lived component ofcolophony; (c) short-lived and (d) long-lived component of Venice turpentine; (e)short-lived and (f) long-lived component of the mixture of sandarac; (g) short-lived

and (g) long-lived component of the mixture of Manila copal. For an easier com-parison fluorescence amplitudes are shown normalised to the maximum value andvalues of �SHORT, �LONG and R are provided. Dashed vertical lines indicate the mainpeak wavelength of each fluorescent component.

respect to that observed in the triterpenic varnishes, which extendsinto the red region of the electromagnetic spectrum in the case ofcolophony and Venice turpentine and towards the green spectralregion in the case of sandarac and Manila and Congo copal. In itsturn the long-lived component shows small differences ascribableto the five materials analysed: a broad and symmetric emissionpeaked at 520 nm for colophony, an emission peaked at 480 nmwith a small shoulder at 420 nm for Venice Turpentine and an emis-sion peaked at 460 nm for sandarac (Fig. 6), at 450 nm for Congocopal and at 470 nm for Manila copal.

3.4. Mixtures of different varnishes

Mixtures of varnishes containing colophony, elemi and sandaracyield different EE spectra than those observed in single varnish sam-ples seen in Figs. 3 and 5, however the EE spectra in mixtures (Fig. 7)may retain features from the constituent materials.

In general, the presence of colophony in mixtures contributesto emissions at longer excitation wavelengths (seen in all threemixtures containing colophony). For example in a mixture ofcolophony:sandarac (Fig. 7a) the maximum emission is observedwith similar excitation wavelength as that seen in colophony, but

292 A. Nevin et al. / Talanta 8

Fig. 7. EE spectra of varnishes made from mixtures of (a) colophony:sandarac, (b)colophony:elemi, (c) elemi:sandarac and (d) colophony:elemi:sandarac.

0 (2009) 286–293

with a Stokes shift of 90 nm, greater than that seen in sandarac andless than that of colophony. The time-resolved analysis of the emis-sion from the mixtures appears to be quite complicated. In fact, itcan happen, as for the case of the mixture of colophony:sandarac,that both the short- and long-lived components of the emissionfrom the mixture are characterised by spectral features which arefound between those observed in the pure varnishes forming themixture, but, in general, it is not possible to easily differentiatefluorescence contributions from mixed varnishes.

In Table 1, the positions of the maximum of the short- and long-lived component of the emission are further summarised.

4. Discussion

While in most cases it is difficult to interpret spectra withreference to specific fluorophores [4], the presence of small butappreciable differences in EE spectra and spectrally resolved fluo-rescence decays is an indication of the chemical variation among thevarnishes analysed, which is ascribed to different sources of materi-als employed. Importantly, despite the dependence of fluorescenceon ageing, spectra of materials reported here are comparable tothose published previously by others [1,4,15].

From an initial examination of the maximum excitation andemission wavelengths as shown in Table 1, shellac is easily dis-tinguished from other varnishes. However only small differencesin the maximum excitation and emission wavelength are found forditerpenoids and triterpenoids; for example mastic and Venice tur-pentine have maxima at (305–10:445–65). Mastic, colophony andVenice turpentine seem to present high Stokes shifts (≥135 nm),significantly greater than the other terpenoids. However, in com-parison with fluorescence excitation and emission maximum, EEspectra indicate further differences in the emission properties ofthe varnishes studied, hence corroborating the limitation of consid-ering only maxima in fluorescence spectra. Using a single excitationwavelength, it may still be possible to better distinguish materialswith the aid of time-resolved fluorescence spectroscopy.

Since materials analysed are characterized by multiple fluo-rophores which emit in different spectral bands, bi-exponentialmodelling of time-resolved data provides a better fit with respectto results obtained with a mono-exponential fit. In detail, in Table 1the mean reduced �2 values for the two exponential models aregiven, where the mono-exponential fit was carried out by consider-ing only the decay following the first 10 ns after excitation. Indeed,fluorescence lifetime analysis of spectral bands provides a betterindication of the presence of emissions from at least two distinctgroups of fluorophores in each material: specifically, the emissionspectrum of the long-lived component can be used to distinguishcolophony from the other terpenic resins, whereas the narroweremission spectrum of the short-lived component of triterpenic var-nishes can be used to distinguish them from diterpenoid media.

However, the resolution of mixtures of varnishes using fluo-rescence spectroscopy indicates that the contribution from onevarnish of the mixture is predominant. As a general observation,in many cases time-resolved and EE spectroscopy are not able toseparate the fluorescent components of mixed materials, but morerefined statistical analyses on spectral and time-resolved data couldbe carried out to address the resolution of mixtures of multiple var-nishes. For example, the combination of colophony:elemi (Fig. 7b)does not yield EE spectra which are easily rationalised, as the posi-tion of the maximum shifts and broadens with respect to that of the

two varnishes. In turn, the EE spectrum of elemi:sandarac (Fig. 7c)does not differ from that of pure elemi in the position of maximumemission, whereas fluorescence intensity at the maximum is moreintense than that of individual varnishes and a change is observedin the shape of the contours. The reason for this phenomenon is

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ot clear; it is possible that both chemical and physical interactionsetween the mixed materials occur or that a stratigraphic separa-ion of the components takes place. In this latter case, the use ofonfocal fluorescence microscopy or other stratigraphic samplingechniques (MALDI) could be investigated.

. Conclusion

The analysis and identification of varnishes using fluores-ence remains a complex problem, which can be addressed,n part, by the integration of excitation emission spectroscopynd complementary time-resolved fluorescence spectroscopy, ashown in this work. Clear differences in EE spectra and short-nd long-lived fluorescence emissions are evident and can bemployed to better understand the materials responsible of themission observed in varnishes. Based on the position of emis-ion maximum, as well as the shape of the contours describinguorescence, it may be possible to classify simple varnishes

nto groups which correspond to their chemical composition:esquiterpenoids and polyesters (shellac), triterpenoids and diter-enoids.

The analysis of mixtures has also been carried out, and revealedhat in some cases various contributions to the observed fluores-ence spectra may be discerned from careful analysis of EE andime-resolved spectra. Further work based on factor analysis (forxample PARAFAC), which has been applied to EE spectra of mix-ures of fluorophores in food [23], may be used to create a moreobust method for the deconvolution of EE spectra of single and

ultiple varnishes. Such analysis could be strengthened by therecise identification of specific groups of fluorophores present inhe complex mixtures of resins employed as varnishes, potentiallychievable with chromatographic techniques.

Instrument varnishes may also be mixed with dyes and col-rants, as well as pigments (for example cinnabar) [14], whichay complicate the interpretation of fluorescence spectra [31]. The

resence of underlying varnish and pigmented layers may alsoompromise the detection of fluorescence; indeed fluorescent orbsorbent substrates (wood or painted surfaces) may also modifyhe observed emission spectrum of materials through scatteringnd reflectance of emitted photons [28]. Therefore, the role ofigments and colorants in the modification or absorption of flu-rescence should be further studied.

Another important factor which has not been directly addressedn this work, but which has been investigated by others [1,4], is thegeing and oxidation of varnishes, which is well-known to producehemical and physical changes [18], both of which may have signif-cant implications for varnish detection using fluorescence; further

ork is therefore necessary on a larger set of naturally aged materi-ls which would better reflect materials found on works of art and

istorical musical instruments.

Finally, within the context of the analysis of both instrumentss well as paintings, fluorescence spectroscopy, while unable torovide the specific and definite identification of materials andarkers, which is afforded by alternative destructive techniques

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0 (2009) 286–293 293

[15], remains an important tool for analysis. Extensively used inpractice by restorers and art historians, fluorescence can indeedprovide indications which go beyond the identification of areas ofrepainting and repairs, and may be used to guide further analyticalinvestigations of musical instruments using selected complemen-tary techniques.

Acknowledgements

Linda Berger is thanked for the preparation of the varnish films.Collaborative work at CUSBO was supported in part by the EuropeanCommission through the Research Infrastructures activity of FP6(“Laserlab-Europe” RII3-CT-2003-506350).

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