Diagnostic Raman spectroscopy for the forensic detection of biomaterials and the preservation of cultural heritage

This paper reviews the contributions of analytical Raman spectroscopy to the non-destructive characterisation of biological materials of relevance to forensic science investigations, including the sourcing of resins and the identification of the biodegradation of art and archaeological artefacts. The advantages of Raman spectroscopy for non-destructive analysis are well-appreciated; however, the ability to record molecular information about organic and inorganic species present in a heterogeneous specimen at the same time, the insensitivity of the Raman scattering process to water and hydroxyl groups, which removes the necessity for sample desiccation, and the ease of illumination for samples of very small and very large sizes and unusual shapes are also apparent. Several examples are used to illustrate the application of Raman spectroscopic techniques to the characterisation of forensic biomaterials and for the preservation of cultural heritage through case studies in the following areas: wall-paintings and rock art, human and animal tissues and skeletal remains, fabrics, resins and ivories.     

Analysis of the Chemical Composition of Red Pigments and Inks for the Characterization and Differentiation of Contemporary Prints

Prints are one of the most popular artistic forms. They consist of an original matrix that is printed on a paper support. The stamps are part of a series, and each series is composed of a particular number of prints. Many contemporary prints are made using oil inks and synthetic pigments (reds and yellows). Inks are mainly composed of pigments (organic or inorganic) and a binding medium. The analysis of inks has the potential to facilitate and complement the identification of stamps of different origins.

Fourier transform infrared spectroscopy (FTIR) and Scanning Electron Microscopy-Energy Dispersive X-ray (SEM-EDX) are techniques that are typically available in museums and centers related to the study of works of art. Both can be classified as micro-destructive and provide complementary information about the organic and some inorganic compounds (FTIR), and the elemental composition (SEM-EDX). In this article, the two techniques were used to analyze the composition of red ink in prints. As a result of these analyses, it was possible to distinguish among nearly all of the pigments and inks, indicating that the composition of the red ink can be reliably used to differentiate between stamps of different origins in a series of prints.     

Spectroscopic investigation of a ‘Virgin of Sorrows’ canvas painting: A multi-method approach

The investigation of unmatched ancient objects is an attentive and arduous activity to conservation scientists. An important aspect of art analysis is the question on sampling and avoiding damage on the artefact during the study. A possible way to maximize the information that is extracted from the historical object is using several sensitive micro-analytical techniques on the same micro samples. As an illustration of this multi-method approach, in this work, a canvas painting ‘Virgin of Sorrows’ was studied and its materials were analysed in order to roughly date and to authenticate this object of art. Proton induced X-ray emission (PIXE), neutron activation analysis (NAA), optical microscopy, scanning electron microscopy (SEM), micro-Raman spectroscopy (MRS) and Fourier transform infrared spectroscopy (FT-IR) were used, obtaining successful results. These methods allowed identifying the different inorganic pigments (iron oxide, carbon black, white lead, Prussian blue) as well as indigo. Optical microscopy and SEM revealed the layered structure of the samples, while FT-IR enabled to determine the nature of the varnish used (shellac). By using these complementary techniques, it was possible to identify the materials in the painting, which are indicative for the period of manufacturing the artwork.     

Characterization of Japanese color sticks by energy dispersive X-ray fluorescence,X-ray diffraction and Fourier transform infrared analysis

This work comprises the use of energy dispersive X-ray fluorescence (EDXRF), X-ray diffraction (XRD) and Fourier transformed infrared (FTIR) techniques for the study of the composition of twentieth century traditional Japanese color sticks. By using the combination of analytical techniques it was possible to obtain information on inorganic and organic pigments, binders and fillers present in the sticks. The colorant materials identified in the sticks were zinc and titanium white, chrome yellow, yellow and red ochre, vermillion, alizarin, indigo, Prussian and synthetic ultramarine blue. The results also showed that calcite and barite were used as inorganic mineral fillers while Arabic gum was the medium used. EDXRF offered great potential for such investigations since it allowed the identification of the elements present in the sample preserving its integrity. However, this information alone was not enough to clearly identify some of the materials in study and therefore it was necessary to use XRD and FTIR techniques.     

Multimethod analysis of Iranian Ilkhanate ceramics from the Takht-e Soleyman palace

The present work describes an analytical study performed on several pieces of Iranian Ilkhanate glazed ceramics from the Takht-e Soleyman palace (Iran, thirteenth century). Several advanced instrumental techniques, including pyrolysis–gas chromatography–mass spectrometry, Fourier transform IR spectroscopy, light microscopy, X-ray diffraction, scanning electron microscopy–X-ray microanalysis and voltammetry of microparticles, were used. The results obtained led to identification of the chemical and mineralogical composition of the pastes and glazes and the colouring agents. Corrosion processes associated with the extreme burial conditions in which the pieces remained for centuries were characterized in some areas of the glazes. A drying oil was identified as the main component of the organic material that was used as the adhesive for the decorative gold sheets applied on the glazes. This finding is in good agreement with traditional recipes. Interestingly, this drying oil exhibits an unusual composition as the gold sheet preserved it from external ageing agents (light, atmosphere, etc.).     

Fourier transform infrared spectroscopy and the analytical study of works of art for purposes of diagnosis and conservation

This paper is a study of the analytical capacity of Fourier transform infrared spectroscopy (FT-IR) for any type of samples from works of art. The analytical information obtained with this technique on organic and inorganic compounds is extremely useful in the preliminary studies necessary for diagnosis and to decide on the conservation process. This paper reports the analysis and study with FT-IR on samples extracted from the microlayers in several works of art from different periods (16th to 18th century) comprising wall paintings and canvas. The most outstanding of these being the frescoes by A. Palomino from two ceilings in the Santos Juanes church. The analytical procedures for the different components of the works studied such as

1. (a) varnish applied to wood panels and canvas paintings,

2. (b) binding media and pigments used by the artists,

3. (c) inerts, mortars, stuccos and grounds have been optimized. The FT-IR technique offers a quick analysis of microsamples (less than 0.5 mg) and is able to characterise the different molecular groups which provide information on the nature of the different materials of organic and inorganic origin used by the artist and thus permits the diagnosis of pathologies requiring conservation treatment.

Characterization of fresh and aged natural ingredients used in historical ointments by molecular spectroscopic techniques: IR,Raman and fluorescence

Natural organic materials used to prepare pharmaceutical mixtures including ointments and balsams have been characterized by a combined non-destructive spectroscopic analytical approach. Three classes of materials which include vegetable oils (olive, almond and palm tree), gums (Arabic and Tragacanth) and beeswax are considered in this study according to their widespread use reported in ancient recipes. Micro-FTIR, micro-Raman and fluorescence spectroscopies have been applied to fresh and mildly thermally aged samples. Vibrational characterization of these organic compounds is reported together with tabulated frequencies, highlighting all spectral features and changes in spectra which occur following artificial aging. Synchronous fluorescence spectroscopy has been shown to be particularly useful for the assessment of changes in oils after aging; spectral difference between Tragacanth and Arabic gum could be due to variations in origin and processing of raw materials. Analysis of these materials using non-destructive spectroscopic techniques provided important analytical information which could be used to guide further study.     

Unilateral NMR, 13C CPMAS NMR spectroscopy and micro-analytical techniques for studying the materials and state of conservation of an ancient Egyptian wooden sarcophagus

A multi-technique approach was employed to study a decorated Egyptian wooden sarcophagus (XXV–XXVI dynasty, Third Intermediate Period), belonging to the Museo del Vicino Oriente of the Sapienza University of Rome. Portable non-invasive unilateral NMR was applied to evaluate the conservation state of the sarcophagus. Moreover, using unilateral NMR, a non-invasive analytical protocol was established to detect the presence of organic substances on the surface and/or embedded in the wooden matrix. This protocol allowed for an educated sampling campaign aimed at further investigating the state of degradation of the wood and the presence of organic substances by ¹³C cross polarization magic angle spinning (CPMAS) NMR spectroscopy. The composition of the painted layer was analysed by optical microscopy (OM), scanning electron microscopy–energy dispersive spectroscopy (SEM–EDS), Raman and surface enhanced (resonance) Raman spectroscopy (SERS/SERRS), infrared and GC–MS techniques, evidencing original components such as clay minerals, Egyptian green, indigo, natural gums, and also highlighting restoration pigments and alteration compounds. The identification of the wood, of great value for the reconstruction of the history of the artwork, was achieved by means of optical microscopy.     

Investigation of nanoorganized biomaterials of marine origin

Naturally occurring nanoorganized biomaterials of marine origin provide an abundant source of novel bone and cartilage replacement materials, and enable the development of novel biomimetic composites. The design of novel biomaterial relies on an understanding of the organic matrices and templating structures. The aim of the present study was to investigate the composition and the properties of skeletal structures of marine sponge (Verongula gigantea) and octocorals (Isidella sp.) in particular by using instrumental analytical (i.e. electron transmission and scanning microscopic methods, vibrational spectroscopies) methods. Modern gentle demineralization techniques were used. It was shown, that the demosponge V. gigantea has much potential as a biomaterial due to the multilayered structure of its rigid fibrous skeletons. The results of FTIR and Raman spectroscopy unambiguously showed that all specimens of the investigated sponge have α-chitin as the main skeletal component. Nano-crystalline aragonite was isolated and identified in V. gigantea, a sponge usually described as lacking a mineral skeleton. Bamboo corals of the Isididae family were additionally investigated. An inorganic component within the deep-sea octocoral Isidella sp. could be clearly identified as calcite by using Raman spectroscopy. The organic part was identified as a nanoorganized fibrillar proteinaceous matrix with acidic properties.     

Chemical signatures of fossilized resins and recent plant exudates

Amber is one of the few gemstones based on an organic structure. Found over most of the world, it is the fossil form of sticky plant exudates called resins. Investigation of amber by modern analytical techniques provides structural information and insight into the identity of the ancient plants that produced the source resin. Mass spectrometric analysis of materials separated by gas chromatography has identified specific compounds that are the basis of a reliable classification of the different types of amber. NMR spectroscopy of bulk, solid amber provides a complementary classification. NMR spectroscopy also can be used to characterize modern resins as well as other types of plant exudates such as gums, gum resins, and kinos, which strongly resemble resins in appearance but have very different molecular constitutions.     

Terracotta polychrome sculptures examined before and after their conservation work: contributions from non-invasive in situ analytical techniques

The potential of non-invasive in situ analytical techniques such as portable Raman, portable X-ray fluorescence, portable optical microscope and fibre optics reflectance spectroscopy has been shown studying painted layers of Renaissance terracotta polychrome sculptures belonging to the statuary of Santo Sepolcro Church in Milan. The results obtained allowed pointing out the contribution of these techniques to the compositional diagnostic, providing complete information, in some cases, better than micro-destructive techniques, on the kind of pigments used on the external painted layers. Moreover, a comparison with the results obtained before the last conservation work (2009) with micro-destructive techniques allowed ascertaining the removal of the external painted layers during the conservation operations.     

Classification and identification of organic binding media in artworks by means of Fourier transform infrared spectroscopy and principal component analysis

Fourier transform infrared spectroscopy is a powerful analytical technique to study organic materials. However, in Cultural Heritage, since the sample under analysis is always a complicated matrix of several materials, data analysis performed through peak-by-peak comparisons of sample spectra with those of standard compounds is a tedious method that does not always provide good results. To overcome this problem, a chemometric model based on principal component analysis was developed to classify and identify organic binding media in artworks. The model allows the differentiation of five families of binders: drying oils, waxes, proteins, gums, and resins, taking into account the absorption bands in two characteristic spectral windows: C–H stretching and carbonyl band. This new methodology was applied in the characterization of binders in three kinds of artworks: papers of historical, archeological, and artistic value, easel paintings, and polychromed stone-based sculptures.     

Novel analytical methods for characterising binding media and protective coatings in artworks

Since the first reported analytical studies and technical examinations of art and archaeological objects conducted in the late 18th century, analytical techniques and methods applied to the study of artworks have constantly grown. Among the materials composing the art object, organic compounds used as binding media or protective coatings have attracted the attention of the conservation profession given their noticeable ability for undergoing morphological and chemical changes on ageing. Thus, the aim of this paper is to review the most recent advances in the identification and determination of organic compounds present in art and art conservation materials. Immunofluorescence techniques have been proposed in recent decades as an alternative to the classical and simpler microchemical tests. Besides, a variety of instrumental techniques have also been improved in an attempt to enhance the sensitivity, repeatability and accuracy of the analytical results. Spectroscopic techniques, such as UV-vis, FTIR and Raman spectroscopy, have been coupled with light microscopes for these purposes. Synchrotron radiation FTIR microspectroscopy has also been successfully applied to the analysis of artworks. Mass spectrometry has also been increasingly used as a detector system coupled with a chromatographic device. Chromatographic methods have also improved in recent years. Paper and thin layer chromatographic techniques have been progressively replaced with gas chromatography (GC), pyrolysis-GC, high performance liquid chromatography and capillary electrophoresis. More complex proteomics hyphenated techniques, such as nano-liquid chromatography-nano-electrospray ionisation/collision quadrupole time-of-flight tandem mass spectrometry, have been recently applied to the identification and determination of proteinaceous binders. Microbeam analytical techniques have also been incorporated into the list of advanced instrumental techniques for art conservation purposes. Finally, a number of new instrumental techniques have been proposed as a suitable alternative to the conventional microscopy techniques for morphological studies.     

Analyses of the surfaces of concrete by Raman and FT‐IR spectroscopies: comparative study of hardened samples after demoulding and after organic post‐treatment

Concrete surfaces were studied by two spectroscopic techniques, FT‐IR (in ATR mode) and Raman, to establish a nondestructive method to analyze the distribution of hydrated and organic phases. The surface composition of ordinary clinker, polished concrete, concrete after demoulding, and coated concrete as used in building construction was studied. The clinker's mineral phases and the polished concrete were first analyzed by Raman spectroscopy to determine a spectrum database of the specific phases located on the surface of the concrete. Then, both spectroscopic techniques were used to analyze, directly, the surface of hardened concrete after demoulding. No impact of roughness or porosity was highlighted using Raman spectroscopy; many cementitious, or hydrated phases (alite, belite, tricalcium aluminate, ferrite, portlandite and ettringite) were clearly identified. FT‐IR in ATR mode only identified some hydrated phases: portlandite and CaO? SiO₂? H₂O (C? S? H), but organic residues from the demoulding oil were characterized by this technique. Furthermore, the convenience of using these techniques together was tested by analyzing the composition of concrete surfaces coated by different organic post‐treatments. FT‐IR spectroscopy was useful to identify the main organic groups at the concrete surface, whereas Raman spectroscopy was especially able to characterize the mineral/hydrated phases under a thick post‐treatment layer (constituted of polyester varnish). Due to their own specificities, these complementary techniques should be used together to easily identify all the mineral phases and organic residues/coatings on concrete surfaces. Copyright © 2010 John Wiley & Sons, Ltd.     

Human exhaled air analytics: biomarkers of diseases

Over the last few years, breath analysis for the routine monitoring of metabolic disorders has attracted a considerable amount of scientific interest, especially since breath sampling is a non-invasive technique, totally painless and agreeable to patients. The investigation of human breath samples with various analytical methods has shown a correlation between the concentration patterns of volatile organic compounds (VOCs) and the occurrence of certain diseases. It has been demonstrated that modern analytical instruments allow the determination of many compounds found in human breath both in normal and anomalous concentrations. The composition of exhaled breath in patients with, for example, lung cancer, inflammatory lung disease, hepatic or renal dysfunction and diabetes contains valuable information. Furthermore, the detection and quantification of oxidative stress, and its monitoring during surgery based on composition of exhaled breath, have made considerable progress. This paper gives an overview of the analytical techniques used for sample collection, preconcentration and analysis of human breath composition. The diagnostic potential of different disease-marking substances in human breath for a selection of diseases and the clinical applications of breath analysis are discussed.     

Characterisation of wax works of art by gas chromatographic procedures

To identify the various natural and synthetic substances used by sculptors at the end of the 19th century, several contemporary reference samples were investigated by high temperature gas chromatography (HT GC) and HT GC-MS. Using specific chromatographic conditions and minimising sample preparation, we could separate, detect and identify a wide range of biomolecular markers covering a great variety of molecular weights and volatilities, with a minimum amount of sample, in a single run. Beeswax, spermaceti, carnauba, candellila and Japan waxes as well as pine resin derivatives, animal fats, paraffin, ozokerite and stearin, used as additives in wax works of art, were chemically investigated. In the case of low volatile compounds, transbutylation was performed. The structure of long-chain esters of spermaceti was elucidated for the first time by HT GC-MS analysis. Such a method was then carried out on 10 samples collected on a statuette of Junon by Antoine-Louis Barye (Louvre Museum, Paris, France) and on a sculpture by Aimé-Jules Dalou (Musée de la Révolution Fran?aise, Vizille, France). The analytical results obtained provide new data on the complex recipes elaborated by sculptors at the end of the 19th century.     

Raman spectroscopy in astrobiology

Raman spectroscopy is proposed as a valuable analytical technique for planetary exploration because it is sensitive to organic and inorganic compounds and able to unambiguously identify key spectral markers in a mixture of biological and geological components; furthermore, sample manipulation is not required and any size of sample can be studied without chemical or mechanical pretreatment. NASA and ESA are considering the adoption of miniaturised Raman spectrometers for inclusion in suites of analytical instrumentation to be placed on robotic landers on Mars in the near future to search for extinct or extant life signals. In this paper we review the advantages and limitations of Raman spectroscopy for the analysis of complex specimens with relevance to the detection of bio- and geomarkers in extremophilic organisms which are considered to be terrestrial analogues of possible extraterrestial life that could have developed on planetary surfaces.     

Micro Raman spectroscopy used for the study of corrosion products on copper alloys: study of the chemical composition of artificial patinas used for restoration purposes

Studying the atmospheric corrosion of copper alloy artifacts is important to acquire a better knowledge about the condition of the object and its possible conservation and restoration. The nature of the formed product, e.g. sulfate, carbonate or chloride, depends on factors such as the amount of polluting elements or humidity but may also depend on the nature of the underlying aesthetic patina, applied by the artist. The composition of the patination solution and the method of patination will both influence the nature of this aesthetic patina, i.e. its chemical composition and morphology. However, although a lot of patination recipes exist, little is known about these patinas as far as composition, structure and ageing is concerned. Therefore, a combination of several surface analytical techniques is required for the complete characterization of patina layers. In this paper, Raman spectroscopy is used to study the characteristics of several patinas obtained on copper following different traditional recipes.