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

Combining SERS and microspectrofluorimetry

with historically accurate reconstructions for the
characterization of lac dye paints in medieval
manuscript illuminations
Rita Castro,a Federica Pozzi,b,c Marco Leonab** and Maria Joo Meloa*
In the present study, a number of dark red microsamples from nine illuminated manuscripts of three important medieval Portuguese
monasteries (St. Mamede of Lorvo, Holy Cross of Coimbra and St. Mary of Alcobaa) were analyzed using a multi-analytical approach
to identify the dyes, fillers, binders and other additional paint components. Historically accurate reconstructions of lac dye paints were
prepared 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 close as possible to the medieval ones and ultimately build
a solid database to support the interpretation of the results obtained from the spectroscopic techniques. Surface-enhanced Raman
spectroscopy (SERS) and microspectrofluorimetry were used here for the first time as complementary techniques to characterize
lac dye paints, whereas Raman microscopy and micro-Fourier transform infrared spectroscopy were employed to detect binders
and fillers. While SERS was able to offer a conclusive molecular fingerprint of lac dye, microspectrofluorimetry provided useful
information 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 publishers web site
Keywords: surface-enhanced Raman spectroscopy; microspectrofluorimetry; lac dye; illuminated manuscripts; Romanesque

Introduction

1172

Lac is part of a resinous cocoon secreted by insects on twigs of

branches of host trees. The dark red resinous raw material is
commonly called sticklac.[1,2] Lac dye, the coloring red substance,
represents only 10% of the entire resin matter, and its main components are laccaic acids A and B; laccaic acids C, D and E are also found
in minor quantities[3,4] Fig. 1. In Fig. S1 (Supporting Information), the
possible acidbase forms of laccaic acid A are shown, as well as two
types of chelating sites in the molecule. When refined, the resin gives
the well-known shellac, which is a complex mixture of monoesters
and polyesters of hydroxyl aliphatic and sesquiterpenoid acids.[5]
Erythrolaccin, also shown in Fig. 1, contributes to the yellowish
orange hue that characterizes the resin.[47]
Lac dye was mainly produced in India, Indochina and south of
China and was already known at around 1500 BC when the ancient
Hindu holy text Atharva Veda was written.[8,9] During the 12th
century, lac was being imported to the western Mediterranean
through trade routes created by the Arabs and Jews.[10] Some of its
earliest Occidental documental descriptions are linked to Portugal,
such as the one from Garcia de Orta, published in Goa in 1563,[11] or
the itinerary descriptions that Jan Huygen van Linschoten wrote after
an expedition to Goa ordered by the King of Portugal, in 1596.[12]
In the field of conservation science, lac dye has been mostly identified in historical textiles.[13] One exception is the work conducted at
the National Gallery (London), where several notable occurrences of
lac dye in paintings were reported.[4] The authors have developed
an extraction method that enables the detection of the lac chromophores by high-performance liquid chromatography with a diode-

J. Raman Spectrosc. 2014, 45, 11721179

array detector (HPLC-DAD).[14,15] Kirby also proposes that, in addition to the laccaic acids, the identification of erythrolaccin in a lac
paint may be considered as a marker for the possible presence of
shellac.[4] Over the years, the characterization of lake pigments
and dyes in works of art has greatly benefited from the advent of
new advanced techniques, such as microspectrofluorimetry and
surface-enhanced Raman spectroscopy (SERS),[1620] as well as from
the development of improved sample pretreatments, such as nonextractive gassolid hydrolysis procedures or mild extraction
methods.[2123] Recent articles have shown that SERS can be used
* Correspondence to: Maria Joo Melo, REQUIMTE-CQFB and Department of
Conservation and Restoration, Faculdade de Cincias e Tecnologia, Universidade
Nova de Lisboa, Campus da Caparica, 2829-516 Caparica, Portugal.
E-mail: mjm@fct.unl.pt
** Correspondence to: Marco Leona, Department of Scientific Research, The
Metropolitan Museum of Art, 1000 Fifth Avenue, New York, NY 10028, USA.
E-mail: Marco.Leona@metmuseum.org

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 Juan
Manuel Madariaga.

a REQUIMTE-CQFB and Department of Conservation and Restoration, Faculdade de

Cincias 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, 111 South Michigan
Avenue, Chicago, IL, 60603, USA

Copyright 2014 John Wiley & Sons, Ltd.

Combining SERS and microspectrofluorimetry with reconstructions

Figure 1. Chemical structures of laccaic acid A, laccaic acids B, C and E (B,

R = CH2CH2OH; C, R = CH2CHNH2COOH; E, R = CH2CH2NH2), laccaic acid D
and erythrolaccin.

successfully to unambiguously identify the main chromophore of

lac dye, laccaic acid A.[1822,24] On the other hand,
microspectrofluorimetry has taken groundwork steps in the characterization of lac dye paints and, in particular, in providing
information on the paint recipe used.[16] For the study of lac dye
paints, SERS and microspectrofluorimetry may be described as
complementary techniques, considering that SERS provides a
conclusive molecular fingerprint for the main chromophore,
whereas microspectrofluorimetry offers valuable information on
the global paint formulation (i.e. the chromophores environment).
In addition, the high sensitivity of microspectrofluorimetry enables
us to use the technique in situ with micrometer-level spatial
resolution, unlike SERS on silver colloids that generally requires
microsampling. Results from both techniques need to be
supported by comparison with a comprehensive spectral database,
in which data from selected historical reproductions must be included. The study of documentary sources and pigment recipes is
also indispensable to properly characterize the formulation of these
paints, and so far, few researchers have exploited it.[4,1416,2527]
Lac dye recipes used to prepare the historically accurate
reconstructions were drawn from various sources dating back to
the period between the 11th and 15th century.[2832] The recipes
were taken from Ibn Badis manuscript, an Arabic treatise on chemical technology for bookmaking dated to c. 1025; Mappae Clavicula,
a well known documentary source of recipes that gathers several
documents from the 8th to 12th centuries; O libro de komo se fazen
as kores (LKFK), a Portuguese treatise from the 15th century; the
Bolognese manuscript from the 15th century; and finally, Strasbourg

manuscript, also from the 15th century. These treatises describe

two main preparation methods, where lac dye is either complexed
with a metal iontypically Al3+ in the form of alumforming a
lake 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 given
in a previous work by some of the authors, based on the results of
micro-Fourier transform infrared spectroscopy (-FTIR) analysis of a
minute sample from Lorvo monastery,[25] Fig. S2 (Supporting Information). We would like to note that throughout the text, the word
carmine will be always used as a hue, that is, an attribute for color
and not as indication for carminic acid. The presence of an organic
chromophore in Holy Cross manuscripts was further confirmed
within a MoLab mission.[33] In the present work, we perform a systematic characterization of lac dye mock-up paints prepared according to medieval recipes and compare the results with data from dark
reds and carmine colors found in manuscript illuminations from the
following Portuguese monasteries: Lorvo, Holy Cross and Alcobaa,
Fig. 2.[16,2527,3335] During the Romanesque period (11th13th centuries), these monasteries had an exceptional cultural productivity,
and the paints used for the illuminations were produced in their
scriptoria, with the best colorants available.[25] Because of the sample
size restrictions and the difficulty in achieving an efficient extraction
of the laccaic acids as a result of the resin crosslinkingeven using
the extraction method developed at the National Galleryit was
not possible to perform HPLC-DAD analysis to characterize the
dye. For this reason, microspectrofluorimetry and SERS were
tested in this work as alternative methods to unambiguously
identify the carmine colors typical of medieval Portuguese
illuminations. In this study, SERS was used for the first time in
the analysis of lac dye reds from medieval illuminations; the data
so obtained were then compared with microspectrofluorimetry
results, thus enabling to validate the latter technique as a robust
analytical tool for the detection of lac dye in situ. Raman and FTIR
spectroscopies were also employed as complementary
techniques to identify binders and fillers.[3537]

Experimental
Historically accurate reconstructions
All reconstructions were carefully prepared with distilled water, reagent grade chemicals and sticklac from Kremer. Each recipe was
repeated several times. The lac paints were applied on parchment

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1173

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

R. Castro et al.
(acquired from the Muse du Parchemin, Rouillon), filter paper and/or
glass slides with parchment glue and/or egg white as binderswhich
were the most likely used in these collections.[25,35] Parchment glue
was prepared as described in LKFK, chapter 40;[30] egg white was
made based on the De Clarea treatise.[38]
Lac reference samples were prepared following documented
sources dating from the 11th to the 15th centuries,[2832] adapted
to laboratory conditions. Two types of recipes will be distinguished
for the purpose of this work, that is, free lac dyes (A) and alumlac
complexes (B):
(A) In the Ibn Badis recipe from chapter six,[28] 5 g of ground lac
was extracted in a 50 ml solution of 0.1 g of sodium carbonate and borax (pH 10), then heated, filtered and heated
again until the ink gained thick consistency; LKFK recipe,
from chapter 13,[30] uses 150 ml of urine filtered in a bed of
quicklime and ashes, in a proportion of 2:1 (pH 910);
10 g of ground lac is added into the solution during heating
and then filtered.
(B) In the Mappae Clavicula,[29] 10 g of sticklac is extracted in
100 ml of heated urine (pH 6); then, 0.6 g of alum (AlK
(SO4)212H2O) is added, and the solution filtered; in the
Bolognese 129 recipe,[31] 150 ml of one-week stale urine is
heated; then, ashes are added (pH 10). Five grams of
ground lac is mixed in the solution and filtered again before
0.6 g of alum is added; Bolognese 130[31] uses 60 ml of 20-day
stale urine (pH 67), 10 g of finely ground lac and 2 g of
alum in the same order but in different quantities (note: this
recipe also uses brazilwood, but we decided not to include it
in this study, as it would make spectral interpretation more
challenging at this stage); Strasbourg recipe named Bright
Paris red[32] starts by leaving 5 g of ground lac in a 50 ml
of lye of ashes (pH 11) overnight. The next day, the solution
is heated and 2 g of alum is added to the mix until the
lake precipitate is obtained. The solution is then filtered
and centrifuged.
In addition, we also used as reference materials for the analytical
approach a set of reconstructions previously made in the framework
of another study.[39] These were mainly prepared by extracting 3.6 g
or 7.2 g of ground sticklac with 30 or 60 ml of different acidic and
basic solutions, respectively. These reconstructions, not based on
medieval documentary sources, were applied on parchment with
parchment glue. For the purpose of this work, these will be referred
as lac 1, lac 2 and so on, as also part of type A free lacs, and lac dye
x0 precipitated, as type B complexed lacs.
For further information on the color of the reconstructions,
consult Table S1 (Supporting Information).
Other reference samples
SERS

1174

Laccaic acids A, B, C and E were collected after HPLC-DAD separation of a 10 3 mol/l H2O : MeOH 70:30 lac dye (Fluka) solution. The
instrumentation and solvent gradient were identical to those
described in the preceding texts, although perchloric acid was
replaced by a 10% aqueous solution of formic acid 99.9% (v/v). Each
laccaic acid was collected separately into a round-bottom flask
according to the retention time (several runs were performed to
collect a sufficient amount of each compound). The so obtained
solutions were rotavapped to remove the methanol and then
lyophilized. HPLC analysis of the samples was subsequently

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performed to ascertain the purity of the compounds collected. After

this, the individual laccaic acids were analyzed by SERS using a
Labram 300 Jobin Yvon spectrometer (exc = 633 nm).
Microspectrofluorimetry

Al3+lac complex applied on filter paper (500 l of 10 3 mol/l lac

dye solution in H2O : MeOH 70:30 complexed with 20 l of 1 mol/l
KAl(SO4)212H2O, at pH = 3.8).
UV-Vis

Lac dye solutions in H2O : MeOH 70:30 prepared to a concentration

of 10 5 mol/l.
Historical samples
Twelve microscopic dark red samples from nine Portuguese illuminated manuscripts dating to the 12th and 13th centuries were
analyzed. These manuscripts were produced in St. Mamede of
Lorvo, St. Mary of Alcobaa and Holy Cross of Coimbra monasteries (So Mamede do Lorvo, Santa Cruz de Coimbra, Santa Maria de
Alcobaa) and are currently preserved in the Arquivo Nacional da
Torre do Tombo (ANTT), Biblioteca Nacional de Portugal (BNP)
and Biblioteca Pblica Municipal do Porto (BPMP), respectively.
Microsamples (weight less than 0.1 g) were available from the
following manuscripts: Lorvo 5, ff.6 and 73v (1183), Alcobaa
238, f.206v (13th c.), Alcobaa 249, f.109v (13th c.), Alcobaa 412,
f.10v (13th c.), Alcobaa 419, f.98 (13th c.), Alcobaa 421, f.202
(13th c.), Alcobaa 446, f.96v (13th c.), Santa Cruz 20, ff. 86 and
191 (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 microchisel from Ted Pella microtools under a Leica KL 1500 LCD microscope, equipped with a 12 objective and a Leica Digilux digital
camera, with external illumination via optical fibers. As for the
microsamples taken from the paint reconstructions, a tungsten
needle was used. Microsamples were typically of 2050 m in
diameter and weight <0.1 g.
Instrumentation
SERS

SERS spectra were acquired from silver colloid using a Bruker

Senterra Raman instrument equipped with a charge-coupled
device detector and a 1800 rulings/mm holographic grating
providing a resolution of 35 cm 1. A 488 nm solid-state laser
was employed as excitation source, with a power of about
0.5 mW at the sample. All spectra were obtained with a single
integration of 30 s, focusing just below the surface of the colloid
drop with an Olympus 20 LMPlanFL long working distance
microscope objective.
Raman microscopy

Normal Raman measurements were conducted using a Labram 300

Jobin Yvon spectrometer, equipped with a HeNe laser operating
at 632.8 nm. Spectra were recorded as an extended scan. The laser
beam was focused onto the samples with a 50 or 100 Olympus
objectives. The laser power at the surface of the samples was
between 4.3 and 0.17 mW.

Copyright 2014 John Wiley & Sons, Ltd.

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Combining SERS and microspectrofluorimetry with reconstructions

FTIR

Infrared spectra were acquired with a Nicolet Nexus spectrophotometer coupled to a Continum microscope (15 objective) with
a MCT-A detector cooled by liquid nitrogen. Spectra were obtained
in transmission mode, between 4000 and 650 cm 1, with a
resolution of 4 cm 1 and 128 or 256 scans. Samples were preliminarily 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.
23002400 cm 1.
Microspectrofluorimetry

Fluorescence excitation and emission spectra were recorded with a

Jobin Yvon/Horiba SPEX Fluorog 3-2.2 spectrofluorometer. Fluorescence spectra were corrected for the wavelength response of the
system. For microspectrofluorimetry analyses, the latter equipment
was hyphenated to an Olympus BX51 M confocal microscope, with
spatial resolution controlled with a multiple-pinhole turret,
corresponding to a minimum 2 m and maximum 60 m spot, with
50 objective. Standard dichroic filters of 500 and 600 nm 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 at
610 nm. Both types of spectra were acquired on a 30 m spot
(pinhole 8) and the following slits set: emission slits = 3/3/3 mm
and excitation slits = 5/3/0.8 mm. The optimization of the signal
was performed for all pinhole apertures through mirror alignment
in the optic pathway of the microscope, following the manufacturers instructions. Spectra were collected after focusing on the
sample (eye view) followed by signal intensity optimization
(detector reading). Emission and excitation spectra were acquired
on the same spot whenever possible. The paint reconstructions
were mainly analyzed in situ, while the historical samples were
analyzed in microsamples. Five spots per sample were measured
to ensure reproducibility of the results.
UV-Vis

UV-Visible absorption spectra of the paint reconstructions and

references in a 1-cm cuvette holder were recorded with a Cary
100 Bio spectrophotometer.
HPLC-DAD

HPLC-DAD analyses were carried out in an analytical Thermo

Electron, FinniganTM Surveyor HPLC-DAD system with a Thermo
Electron, FinniganTM Surveyor LC pump, Autosampler and PDA
detector and using a reversed-phase RP18 analytic column
(Nucleosil C18, 250 4.6 mm, 300 5 m) kept at controlled
temperature (35 C). Samples were injected into the column via a
Rheodyne injector with a 25 l loop. The elution gradient used at
a flow rate of 1.7 ml/min consisted of A: HPLC-grade methanol
and B: 0.3% (v/v) perchloric acid in Millipore ultrapure water. The
gradient elution program was the following: 02 min, isocratic 7%
A; 28 min, linear gradient to 15% A; 825 min, linear gradient to
75% A; 2527 min, linear gradient to 80% A; 2729 min, linear to
100% A; and 2940 min, isocratic 100% A.[40]

were used as SERS substrate. A detailed description of the synthesis

is reported elsewhere.[18]
SERS analysis was performed after deposition of 0.8 l of the Ag
colloid and 0.1 l of 0.5 mol/l KNO3 aqueous solution onto each
microsample. Spectra were collected by focusing the laser beam
onto the microaggregates that formed inside the dye-colloid
droplet a few seconds after the deposition of the Ag nanoparticles
and KNO3. Several spectra were acquired continuously until the
droplet dried out.
Three sorts of SERS procedures were used, according to the type
of sample:
(1) For free lac reproductions (type A recipes), SERS analysis
was performed directly on the microsamples without
any pretreatment;
(2) For lacalum reproductions (type B recipes), a non-extractive
gassolid hydrolysis pretreatment was used, in which the
microsamples are exposed to hydrofluoric acid (HF) vapor
in a closed microchamber for 5 min. This procedure aims to
hydrolyze the dyemetal complex and increase the analyte
adsorption on the nanosized metal substrate, thus enhancing the SERS signal.[21]
(3) For the historical samples, because it was possible to have
the dye as free lac dye or as lac lake pigment, a two-step
procedure was followed by analyzing the sample first without 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 be
discussed 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 are
described in the treatises: (A) free lac dyes, characterized by the absence of a complexing metal ion (described in Ibn Badis and LKFK);
(B) lac lake pigments, obtained by the addition of alum (described
in Bolognese, Mappae Clavicula and Strasbourg). Non-complexed
lac paints tend to be more reddish and glossy (with a pH close to
neutral), 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 or
obscure instructions, such as omitted measured quantities or
heating temperatures, which makes them open to interpretation.
Bolognese, Strasbourg and LKFK recipes are perhaps the ones that
need more extensive interpretation work, because of deficient
information (for example, Bolognese and Strasbourg lack
specific information on the quantities of alum, whereas LKFK,
written in Hebrew, poses linguistic challenges that may affect
its translation). As for the Mappae clavicula and Ibn Badis,
recipes are significantly more detailed, which makes them
easier to reproduce accurately.
FTIR and Raman results

Colloid synthesis, SERS methodology and sample pretreatments

J. Raman Spectrosc. 2014, 45, 11721179

The dark reds (carmine) and pink colors found in the manuscript
illuminations 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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1175

Silver nanoparticles prepared by microwave-supported glucose

reduction of silver sulfate with sodium citrate as capping agent

R. Castro et al.
white lead and vermilion (by Raman, infrared and microfluorescence
UV-Vis and supported by XRF[25,33,34]); some darker reds in Holy Cross
manuscripts were identified as the dye admixed with vermilion,
Fig. S4 (Supporting Information).[25,34] All of these paints were
applied with a proteinaceous binder, such as parchment glue or
egg white.[35] For these carmine paints, the FTIR spectra essentially
display the binders fingerprint as well as, in most cases, the signals
as a result of the fillers, Fig. S4 (Supporting Information). In some
samples, lac dye is indirectly detected upon identification of the
CH stretching absorption bands of the shellac resin, Fig. S2
(Supporting Information). Chalk or gypsum was most commonly
detected for Alcobaa samples but was also found in a few
specimens from Holy Cross. Because of the high fluorescence in
the Raman signal, these fillers were only detected by FTIR.
SERS results
Historically accurate reconstructions and standard laccaic acids
SERS spectra for the lacalum reconstructions could only be
obtained 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 circa
1578, 1464, 1368, 1326, 1287, 1227, 1188, 1098, 1052, 1010, 453 and
408 cm 1 (Fig. 4(d)). On the other hand, reference SERS spectra
from laccaic acids A, B, C and E as collected from HPLC are shown
in Fig. S5 (Supporting Information). The similarity between the
spectra of laccaic acid A and the commercial mixture (Figs S5a
and S5b) indicates that SERS is detecting primarily the former
compound, that is, the main dye chromophore. We experimentally
acquired the relative composition of lac dye chromophores (from
Fluka), 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 acidic
conditions. This is in agreement with Caamares et al.[24] who found
that the SERS intensity for lac dye increases going from alkaline to

Figure 4. SERS spectra of the historical samples, at exc = 488 nm: (a)
Lorvo 5, f.6; (b) Santa Cruz 20, f.191; (c) Alcobaa 446, f.96v; (d) laccaic
acid A at pH = 2.

acidic pHs. This is likely because of the increase in the resonance Raman effect combined with a lower electrostatic repulsion between
laccaic acid and the negatively charged nanoparticle surface
(capped by the citrate ions) at acidic pH. Within this group of lac
lakes, minor shifts in wave number and slight changes in relative
intensities were observed, which may be attributed to pH effects
or to other components present in the matrix that may interfere
with the SERS signal.
Overall, a clear distinction was observed between the lac dyeAl3+
complexes analyzed after HF treatment and the free lac reconstructions examined without hydrolysis. The latter displayed intense
signals at circa 1622, 1568, 1534, 1460, 1346, 1192, 1100, 1058 and
1011 cm 1, as shown in Fig. 3. As expected from the literature,[24]

3+

1176

Figure 3. On the left, SERS spectra of Al lac complex reconstructions, at exc = 488 nm: (a) Mappae Clavicula; (b) Strasbourg; (c) Bolognese 130; (d) Bolognese
129. On the right, SERS spectra of non-complexed lac reconstructions, at exc = 488 nm: (e) Ibn Badis 2; (f) Libro de komo se fazen as kores; (g) lac dye 3
reconstruction (pH 3); (h) sticklac (raw material).

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Combining SERS and microspectrofluorimetry with reconstructions

in samples where the HF treatment was applied (acidic media) the
band at 1464 cm 1, assigned to CC I ring stretching modes and to
C8OH and C5OH bending modes, displayed the highest intensity.
On the other hand, in the samples analyzed without HF (neutral to
basic media), the highest intensity was observed at 134050 cm 1.
In the literature, this latter band is normally assigned to the deprotonation of the C5OH bending mode in the molecule, Table S2
(Supporting Information).[22,24]
Historical samples
As far as the historical samples are concerned, while regular SERS
proved unsuccessful as a result of interference of the citrate ions
capping the nanoparticle surface, the use of the HF pretreatment
ensured certain dye identification in all cases. This approach
enabled us to confirm that the dark red used in Portuguese
Romanesque illuminations was based on lac dye, Fig. 4 and Table
S3 (Supporting Information). The fact that SERS only worked after
acidic treatment suggested that the historical paints from the
manuscripts 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 without
HF hydrolysis. Overall, the SERS spectra of the historical samples
are very similar to the lake reproductions spectra, revealing only
slight variations in the 15801010 cm 1 region. Interestingly,
Mappae Clavicula (Fig. 3(a)) and Bolognese type B reconstruction
(Fig. 3(c) and 3(d)) spectra showed an excellent resemblance with
the spectrum obtained from Lorvo 5, f.6 historical sample (Fig. 4(a)).
Microspectrofluorimetry results

J. Raman Spectrosc. 2014, 45, 11721179

3+

Figure 5. Excitation and emission spectra from (a) the Al lac complex
.
5
() on filter paper; and its absorption spectra ( ) in solution (10 mol/l);
(b) Lorvo 5, f6 () with Mappae Clavicula recipe (---); (c) Alcobaa 412,
f.10v () with Ibn Badis recipe (---), respectively.

526 nm displays the highest intensity. The emission spectrum, not

so finely resolved, displays a maximum at 593 nm, Fig. 5(a).
When comparing the references discussed in the preceding texts
with the historically accurate reconstructions (Table 1; refer to Fig.
S7 (Supporting Information), for representative fluorescence
spectra), the Bolognese 130 recipe is very similar to the Al3+lac
complex solution described previously, with a slight shift in the
emission spectra. In Mappae Clavicula, the main features in both
emission and excitation spectra are maintained, although in the

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1177

As the need of HF pretreatment for all of the historical samples

examined suggested the use of lacalum pigments in the
manuscript paints, in this section, special attention will be devoted
to reconstructions based on recipes in which lac dye is complexed
with Al3+ ions. Before examining the paint reconstructions though,
the main spectral features of raw sticklac and shellac resin will be
discussed. Figure S6 (Supporting Information) shows the emission
and excitation spectra of the two materials. In the spectra of raw
sticklac, both the resin and all the lac chromophores are detected,
whereas for shellac, the main contribution observed is that of the
resin itself. The laccaic acid chromophores dominate the spectra
for the raw material, and the excitation spectrum is characterized
by a broad, unresolved band, with a maximum at 505 nm. Note that
in all excitation spectra presented, showing the absorption of the
emitting species, for very low intensities, the band at circa 420 nm
is an instrumental artifact. At 490 nm excitation, the emission
spectrum of sticklac mainly displays features of the laccaic acids.
The shellac resins excitation spectrum displays a band at 463 nm,
which compares well with the absorption spectrum published in
literature for erythrolaccin,[4] characterized by maxima at 264, 294,
464 nm, of which only the latter can be detected with our
instrumental setup (dichroic filter 600 nm).
On the other hand, the Al3+lac dye complex solution applied to
filter paper, at pH = 3.8, displays well resolved spectral features, with
a small Stokes shift, indicating that the same species is absorbing
and emitting in the excited state (Fig. 5(a)). The excitation spectra
are characterized by two bands, with maxima at 526 and 562 nm,
in agreement with the absorption spectra. The relative intensities
of the two emission bands may change according to the pH, and
minor shifts may occur depending on the amount of alum present.
Close to neutral and basic pH, the signal is lower and the band at

R. Castro et al.
Table 1. Fluorescence emission and excitation maxima for the main paint reconstructions
Ibn Badis

Mappae Clavicula

LKFK

Bolognese

Strasbourg

589
472

590
520

No signal
No signal

589
526, 562

587
526, 562

em/nm
exc/nm

Table 2. Fluorescence emission and excitation maxima for representative manuscripts

em/nm
exc/nm

Lv 5

ALC 238

ALC 249

ALC 412

ALC 419

ALC 421

ALC 446

SC 20

SC 21

58789
52326

563
522

589
554

587
474

587
556

593
470

593
476

58789
553

58991
520

excitation spectra, the bands are not resolved and the relative
intensities inverted, being the band around 520525 nm the most
intense. The shoulder at circa 470 nm may include the contribution
of 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 is
located at 472 nm. This may be ascribed to the presence of the
erythrolaccin as well as to the complete absence of alum. Given that
in the literature, the detection of erythrolaccin is usually associated
with the presence of shellac resin, we may conclude that
microspectrofluorimetry is indirectly sensing the presence of the
resin and indicating that the latter was incorporated in the final
pigment.[4] To confirm this hypothesis, HPLC-DAD was performed
on the lac reproductions. While the chromatogram of Bolognese
130 reconstruction showed only the main complexed laccaic acid
chromophores at 490 nm, the Ibn Badis was found to contain resin
as well, Figs S8 and S9 (Supporting Information), in accordance with
the microspectrofluorimetric data.
The emission and excitation maxima for the original medieval
samples are displayed in Table 2. The emission spectra show a band
around 589 nm, which is detected consistently for most of the lac
reproductions. As observed in the reconstructions, more pronounced shifts are observed for the excitation spectra. In addition,
some of the original samples display a band at 472 nm, particularly
prominent in the Alcobaa samples, which, as discussed in the
preceding texts, is likely because of the presence of higher amounts
of resin. In terms of signal intensity, more pronounced emissions
are observed in samples from Lorvo (circa 2), whereas spectra of
Alcobaa samples are characterized by lower intensities. When
comparing these with laccaic acids, lacAl3+, sticklac and shellac
spectra, it is possible to conclude that the original samples are
composed of laccaic acids and shellac, Figs 5 and S6 (Supporting
Information). Compared with the reconstructions, the most significant similarities were detected between the historical samples and
Mappae Claviculaalso in accordance with the SERS resultsand
Ibn Badis mock-ups (Fig. 5(b) and 5(c)). The color of these two reconstructions is also very similar to that observed in the manuscripts.

Conclusions

1178

In this work, for the first time, lac dye was unequivocally identified
in medieval illuminations. SERS proved to be an advantageous
technique for the identification of dyes in illuminated manuscripts,
because of the minute sample size required for analysis (1020 m).
By using the unambiguous molecular fingerprint of the dye

wileyonlinelibrary.com/journal/jrs

provided by SERS, we were able to assess the microspectrofluorimetry results and validate this technique as a robust analytical
tool for the detection of lac dye in situ. Work is in progress to further
explore the applications of microspectrofluorimetry.
The availability of historically accurate reconstructions was essential for this study, as it enabled to assemble a consistent spectral
database of mock-ups prepared according to historical recipes, to
be used for comparison in the analysis of samples from actual medieval illuminations. Additionally, comparison with paint reconstructions prepared in the lab shed new light on the fact that the
color shades observed in the medieval illuminations (pink, red
and violet, to brownish hues) may be the result of the processing
of 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 describes the chromophore in its environment. In this sense, these
two techniques, combined in this study for the first time, can be
seen as complementary. The microspectrofluorimetric data obtained so far suggest that it is also possible to detect the resin in
a lac sample indirectly by using erythrolaccin as a marker; this
was further confirmed by HPLC-DAD.
As for future research, we intend to explore the use of mixtures in
lac paints, such as fillers (gypsum, chalk), vermilion, lead white and
several dyes, in order to establish correlations with the historical samples. To elaborate the spectral information obtained by SERS and
microfluorimetry and ascertain whether it may carry details on the
process used to prepare the pigment or other hidden patterns and
features, a multivariate analysis approach will be followed.
In conclusion, our research sheds new light on the use of this
historical dye. Its systematic application in Portuguese medieval
manuscripts 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, in
1498). There is no evidence, to date, that lac dye was being used
by medieval monasteries other than those cited in this work. Thus
far, it has only been identified by SERS in a French polychrome
wood sculpture[18] and in a Spanish crucifix,[22] both dating to
11501200. These three occurrences, in the Iberian Peninsula and
Provence, confirm the existence of an Arabic and Jewish trade
network in that region during the Romanesque period.
Acknowledgements
This work has been financially supported by Portuguese funds
through FCTFundao para a Cincia e a Tecnologia under the
projects PTDC/QUI-QUI/099388/2008 and PTDC/EAT-EAT/104930/

Copyright 2014 John Wiley & Sons, Ltd.

J. Raman Spectrosc. 2014, 45, 11721179

Combining SERS and microspectrofluorimetry with reconstructions

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 Pblica Municipal do Porto (BPMP) for their generous support and collaboration.

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Supporting information
Additional supporting information may be found in the online
version of this article at the publishers web site.

1179

J. Raman Spectrosc. 2014, 45, 11721179

Copyright 2014 John Wiley & Sons, Ltd.

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