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Microspectrofluorimetry and Chemometrics for the Identification of Medieval Lake Pigments

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Microspectrofluorimetry and Chemometrics for the Identification of Medieval Lake Pigments Nabais et al. Herit Sci (2018) 6:13 https://doi.org/10.1186/s40494-018-0178-1 RESEARCH ARTICLE Open Access Microspectrofuorimetry and chemometrics for the identifcation of medieval lake pigments Paula Nabais1 , Maria J. Melo1* , João A. Lopes2*, Tatiana Vitorino1,3, Artur Neves1 and Rita Castro1 Abstract Microspectrofuorimetry ofers high sensitivity, selectivity, fast data acquisition, good spatial resolution (down to 2 μm), and the possibility of in-depth profling. It has proved to be a powerful analytical tool in identifying dyes and lake pigments in works of art. To maximize the extraction of the information present in fuorescence emission and excitation spectra, we propose a chemometric approach to discriminate dark reds to pink colours based on brazil- wood, cochineal, kermes and lac dye. These range of hues was obtained using a diverse range of medieval recipes for brazilwood, kermes and lac colourants and Winsor and Newton archive for cochineal lake pigments; the lake pigments were analyzed as colour paints (arabic-gum and glair were the medieval binders selected). Unsupervised (HCA & PCA) and supervised (SIMCA) modelling were tested, allowing to explore similarities between colourants and classify the spectral data into the diferent lake pigments classes. It was possible to separate the four diferent chromo- phores based on their excitation spectra or bringing together the emission and excitation spectra. The frst method could also diferentiate between the cochineal lake pigments, in particular between crimson lakes with diferent alu- minates and an extender (gypsum) and between carmines with diferent complexing ions (aluminum and calcium). Keywords: Medieval manuscripts, Dyes, Lake pigments, Spectrofuorimetry, SIMCA, HCA, PCA Introduction be available from works of art. Microspectrofuorimetry In the past few years we have been particularly interested ofers high sensitivity and selectivity combined with good in the development of methodologies that will promote a spatial resolution and the possibility of in-depth profling. complete characterization of the organic colourants used It can also be used in situ without any contact with the in the past as well as their degradation products [1–12]. sample or work of art to be analyzed, for movable objects Changes in pigments, whether used pure or admixed, that can be transported in the laboratory [13, 14]. Te can alter the appearance of a painting signifcantly; con- importance of sensitivity is clear when the following facts sequently, the identifcation and state of degradation of are considered: some of the dyes used in the past to create colourants is of fundamental interest, since it provides bright colours may have faded or may have been applied critical information about the artists’ aesthetic perspec- as very thin coats over, or mixed with, an inorganic pig- tive, conceptions and choices, and how the work has ment or extender, and therefore they may be present in changed over time. Terefore, it is desirable to develop very low concentrations. Te possibility of in situ analysis methods that can characterize these materials directly of ancient colourants is a considerable advantage, par- on the artwork, in situ, or from small samples that may ticularly when considering that the techniques currently employed for dye analysis (HPLC–DAD-MS, microFTIR *Correspondence: mjm@fct.unl.pt; jlopes@f.ulisboa.pt and SERS) require micro-sampling [15–17]. Microspec- 1 DCR and LAQV‑REQUIMTE, Faculty of Sciences and Technology, NOVA trofuorimetry also presents some drawbacks, namely the University of Lisbon, 2829‑516 Caparica, Portugal absence of a molecular fngerprint as disclosed in infra- 2 iMed.ULisboa‑Research Institute for Medicines, Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649‑003 Lisbon, Portugal red spectra. Tis limitation may be overcome by com- Full list of author information is available at the end of the article bining surface-enhanced Raman spectroscopy (SERS) © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Nabais et al. Herit Sci (2018) 6:13 Page 2 of 11 and fber-optics refectance spectroscopy in the visible identifcation by an analytical technique such as micro- (FORS) and by using a consistent database build up with spectrofuorimetry very challenging. historically accurate reproductions of references for col- Brazilwood has been extensively found in books of ourants, binders and colour paints, which are the result hours from the 15th–16th c., and in the Galician-Portu- of research into written sources of medieval techniques guese Ajuda songbook, possibly dated from the 13th c. [13, 14, 17]. Tey are part of reproducing the process [5, 11, 12] and is extracted from a tree, Caesalpinia sap- described in the source material as well as molecular pan or other brazilwood species brought to Europe from identifcation and comparison with the original colours. Brazil from the 16th c. onwards (Caesalpinia echinata, Tis leads to a virtuous feedback loop, where reference Caesalpinia brasiliensis, Caesalpinia violacea, Caesal- compounds are validated against originals and are used pinia crista, and Haematoxylum brasiletto) [18]. Kermes to improve the analytical methods applied when identify- was obtained from a small insect, Kermes vermilio, found ing materials [11, 18–22]. A hypothesis that we will test in the kermes oak, Quercus coccifera L. Other important in this work using a chemometric approach. historical sources of red derived from the resin secreted We will focus on four natural red dyes, and their lake from the female lac insect, Kerria lacca, from which pigments, used during the Middle Ages (found in medi- are obtained both the lac dye and the shellac resin. It eval manuscripts and described in technical treatises): lac was applied as a dark red or pink colour in Portuguese dye, kermes, cochineal and brazilwood, Table 1. Te lat- manuscripts and it is characteristic of the Romanesque ter is a favonoid, but the other three are anthraquinone monastic production (12th–13th c) [1, 10]. In the 16th reds extracted from animal sources, which makes their c. most of these sources were replaced by the red and Table 1 The four red colourants studied in this work, with the respective chromophores, provenience and chronology of occurrences in the Mediterranean world (in artworks) Colourant Main chromophore Provenience Chronology Brazilwood Asia From 13th Caesalpinia spp. South-America From 16th Haematoxylum brasiletto Brazilein Kermes Mediterranean BC-15th c. Kermes vermilio Morocco Algeria Kermesic acid Cochineal South-America From 15th Dactylopius coccus Eastern Europe Porphyrophora spp. Carminic acid Lac dye India From 12th Kerria lacca Indochina South of China Laccaic acid A Nabais et al. Herit Sci (2018) 6:13 Page 3 of 11 scarlet colours of the American cochineal, Dactylopius process, performed hierarchically, and depending on coccus, commercialized by the Spanish empire [23]. Simi- the selected algorithm, multiple clustering options are lar species were already found in Eastern Europe, Porphy- possible. Results are typically represented graphically in rophora polonica and Porphyrophora hamelli, known as the form of a dendrogram, where samples are visualized Polish cochineal and Armenian cochineal, respectively according to their similarity [27]. Te SIMCA model, is a [23, 24]. supervised classifcation method. It is based on the devel- In previous publications, we proved that confocal opment of multiple PCA models, each built considering microfuorescence is a powerful tool for in situ analysis samples of a known class or group [28, 29]. Te goal is of colourants based on natural dyes [13, 14, 25]. Natural to allow for classifcation by presenting unknown sam- dyes may be described as weak to medium emitters. Fol- ples to the diferent PCA models composing the SIMCA lowing light absorption, an excited molecule is formed, model. When projecting samples to this model, they are and this fuorophore may lose its excess energy by emit- classifed according to their similarity with the diferent ting light. In a spectrofuorimeter, exciting at a single PCA class models (typically Hotelling’s T2 and squared excitation wavelength and recording the fuorescence in residuals statistics are used to evaluate the distance to the fuorophores’ emission wavelength range results in an each model). Indeed, when projecting one sample, difer- emission spectrum. It is also possible to excite at diferent ent outcomes are possible: (1) the sample might be clas- wavelengths, following the colourant absorption spec- sifed according to one class; (2) the sample is classifed trum, collecting at a single wavelength, obtaining thus an as belonging to two or more classes; (3) the sample is not excitation spectrum that may reproduce the absorption classifed in any of the model’s classes. Tis allows for the spectrum [26]. coverage of high class variability by the principal compo- Te simultaneous acquisition of emission and excita- nents calculated individually,
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