Studies on paint cross sections of a glass painting by using FT‐IR and Raman microspectroscopy supported by univariate and hierarchical cluster analyses

Fourier transform infrared (FT‐IR) and Raman spectroscopy is used for the non‐destructive analysis of painting materials and ageing compounds in micrometric cross sections of a glass painting. The combination of both techniques in conjunction with imaging/mapping function provides the spatial distribution of chemical components identified in vibrational spectra. The aim of our work is to show the applicability of the FT‐Raman mapping technique in the detection of painting materials. We also compare Raman information gained by using two laser excitations at 532 and 1064 nm implemented in microspectrometers with different confocality and spatial resolution. In turn among FT‐IR imaging techniques, we compare chemical images recorded in external reflection and attenuated total reflection modes that give chemical images of different size and spatial resolution. Our FT‐IR and Raman imaging characterize a number of painting materials such as pigments, binders, fillers as well as degradation products. Raman maps are constructed by using the univariate analysis. In turn, a profile of IR images requires the use of a more complex methodology. Here, we compare FT‐IR images of the painting cross sections obtained by using the univariate and hierarchical cluster analysis. We clearly show that the multivariate approach is a powerful tool for the credible construction of IR images, providing the relevant chemical information on the multicomponent stratigraphy of the samples. Moreover, the combination of all the methods allows us to demonstrate their degree of utility for the study on the paint cross sections of the works of art. Copyright © 2013 John Wiley & Sons, Ltd.     

The first in situ Raman spectroscopic study of San rock art in South Africa: procedures and preliminary results

Southern Africa has a rich heritage of hunter‐gatherer, herder and farmer rock art traditions made by using both painted and engraved techniques. Until now, there have been only a handful of studies on the chemical analysis of the paint, as all previous types of analysis required the removal of pigment samples from the sites a practice which has been avoided. Raman spectroscopy is an ideal techniques to analyse the paint non‐destructively and also offers the possibility of in situ work with portable instruments. This article describes the procedures and reports the preliminary results of the first in situ Raman spectroscopic study of rock art in South Africa (also a first worldwide), where we, first, evaluate the capability of a Raman portable instrument in very difficult conditions, second, analyse the paints in order to contribute to a better knowledge of the technology used and, third, evaluate the possible contribution of in situ analyses in conservation studies. The paintings from two different rock art sites were studied. The instrument proved to be highly suitable for in situ analyses in physically very challenging conditions. Most of the pigments and alteration products previously detected under laboratory conditions were identified, thereby giving information on both the pigments and conservation state of the paintings. A layered structure of alteration products and pigment was identified in situ for the first time by controlling the laser power, thereby obtaining the same results as in mapping experiments of cross sections of paint. Copyright © 2010 John Wiley & Sons, Ltd.     

Raman spectroscopic evaluation of the influence of Pseudomonas bacteria on aflatoxin B1 in the BioArena complex bioautographic system

The mechanism of the antibacterial‐toxic effect of aflatoxin B1 (AFB1) was investigated in the BioArena complex bioautographic system that consists of planar liquid chromatographic development and biological detection. In this system, the killing‐inhibiting activity of AFB1 against Pseudomonas savastanoi (Psm) bacterial cells was visualised. The role of formaldehyde (HCHO) was suggested in the antibacterial‐toxic action of AFB1. Raman spectroscopy (RS) was used to investigate whether the excess HCHO found earlier in the AFB1 spot (comparing with background) originated partly from the demethylation of the toxin, or only from the enhanced demethylation of the normal cell ingredients because of the stress situation. Fourier transform (FT) Raman and surface‐enhanced FT‐Raman spectra were obtained in situ about the AFB1 chromatographic and background spots in bacteria‐free and inoculated thin layer chromatography (TLC) layers, and ex situ about their dried methanolic extract. The reduction of the δ CH₃ band of AFB1 (1386 cm⁻¹) in the presence of Psm can indicate the demethylation of aflatoxin at its methoxy‐group in BioArena. It seems from the Raman spectra that, apart from demethylation, aflatoxin does not suffer other structural changes. However, Psm can reduce the Raman activity of νCO bond of the pyran ring of AFB1, causing lower intensity and a broader band (1747 and 1754 cm⁻¹). Psm can also disrupt the strong interactions between the AFB1 and the adsorbent layer through the νCO bond of the cyclopentene ring causing a blue‐shift (1667 → 1686 cm⁻¹). The very reactive HCHO that originated from exogenous sources, e.g. from the demethylation of AFB1, has no determined biochemical pathway, so it means higher risk. Copyright © 2008 John Wiley & Sons, Ltd.     

Use of a handheld Raman spectrometer for fast screening of microbial pigments in cultures of halophilic microorganisms and in microbial communities in hypersaline environments in nature

A compact handheld Raman spectrometer equipped with a 532 nm excitation laser was used to detect pigments as possible biomarkers in autotrophic (cyanobacteria and purple sulfur bacteria) and heterotrophic halophilic microorganisms (Archaea of the family Halobacteriaceae, Salinibacter). Common as well as less common carotenoids, including α‐bacterioruberin, salinixanthin, and spirilloxanthin derivatives were detected in cell pellets of model organisms belonging to the genera Haloferax, Haloarcula, Halobacterium (Archaea), Salinibacter (Bacteroidetes), and Ectothiorhodospira (Gammaproteobacteria). Direct measurements on such cultures provide fast and reliable identification of pigments. Bacterioruberin was detected as the dominant carotenoid in pellets of cells collected from the saltern crystallizer ponds in Eilat, Israel. Raman analysis of the colored layered microbial communities in a benthic gypsum crust in the saltern evaporation ponds showed signals consistent with the presence of myxoxanthophyll and echinenone carotenoids in the upper orange and dark‐green cyanobacterial layers. Chlorophyll a and phycocyanin expected in the green layer were not detected using the green excitation. Spirilloxanthin dominates the red layer below, inhabited mainly by purple sulfur bacteria. To our best knowledge, this is the first attempt to detect and identify pigments in natural microbial communities consisting of different types of halophilic microorganisms by direct Raman spectrometric measurements using light compact handheld devices. Copyright © 2013 John Wiley & Sons, Ltd.     

FT‐IR and FT‐Raman investigations of the chemosensing material para‐hexafluoroisopropanol aniline

As an important chemosensing material involving hexafluoroisopropanol (HFIP) for detecting nerve agents, para‐HFIP aniline (p‐HFIPA) has been firstly synthesized through a new reaction approach and then characterized by nuclear magnetic resonance and mass spectrometry experiments. Fourier transform infrared absorption spectroscopy (FT‐IR) and FT‐Raman spectra of p‐HFIPA have been obtained in the regions of 4000–500 and 4000–200 cm⁻¹, respectively. Detailed identifications of its fundamental vibrational bands have been given for the first time. Moreover, p‐HFIPA has been optimized and vibrational wavenumber analysis can be subsequently performed via density functional theory (DFT) approach in order to assist these identifications in the experimental FT‐IR and FT‐Raman spectra. The present experimental FT‐IR and FT‐Raman spectra of p‐HFIPA are in good agreement with theoretical FT‐IR and FT‐Raman spectra. Copyright © 2009 John Wiley & Sons, Ltd.     

Precious coral non‐destructive characterization by Raman and XRF spectroscopy

Precious corals are some of the most valuable living marine resources, growing and commercially exploited only in limited areas of the world, namely the Mediterranean Sea and the Northern Pacific Ocean. Their skeleton is formed by calcium carbonate crystallized in the form of calcite whereas their color is because of the presence of partially demethylated polyene pigments. Recently, Pacific corals have been included in the appendix II of CITES list, while Mediterranean corals are still excluded. Different Corallium species of Corallidae family (e.g. Corallium rubrum, Corallium elatius and Corallium secundum) collected from different locations of the Mediterranean Sea and the Pacific Ocean were analyzed by Raman spectroscopy for the characterization of the reddish pigment and by X‐ray fluorescence (XRF) for the determination of the chemical composition of their skeletons, in order to obtain molecular and elemental data with two relatively easy and non‐destructive techniques, which can be used quite steadily for authentication purposes. Raman analysis demonstrated the presence of specific vibrational bands useful to identify the colored pigments as a mixture involving methylated and demethylated polyenes such as carotenoids and parrodienes, characterized by the presence of ―CH₃ groups along the polyene chain. The ratio between the Raman signal and fluorescence background was found to vary as a function of the macroscopic color of the coral, but Raman analyses resulted inadequate for distinguishing between corals having similar color but different origins. On the other side, XRF data provided reliable information for an appropriate separation between Pacific and Mediterranean corals at the elemental level. The results of this study will be of great relevance for the authentication and identification of the origin of corals in trade market by means of completely non‐destructive techniques. Copyright © 2016 John Wiley & Sons, Ltd.     

Raman spectroscopy as a tool to the in situ study of three lichens species from Antarctica and Brazil

In this investigation the chemistry of the lichens Gondwania regalis, Teloschistes exilis and Xanthoria candelaria (Teloschistaceae) have been recorded by means of Raman spectroscopy. The non‐destructive analysis provided the recognition of parietin and conjugated polyenes, probably belonging to the carotenoid family for all the investigated specimens. Bands at ca. 1370 and 1600 cm⁻¹, respectively, assigned to the ν(C―O) and ν(CO) modes of the phenyl group of the anthraquinone compound, as well the bands at ca. 1005, 1158 and 1527 cm⁻¹, possibly assigned to the β‐carotene in the FT‐Raman spectra, have provided valuable spectroscopy data for the identification of the biomarkers for these lichen pigments. Thus, this is the first report of parietin and carotenoid in T. exilis and X. candelaria tissues even as the parietin anthraquinone for G. regalis tissues, which are effective pigments against free radicals from UV radiation. Copyright © 2014 John Wiley & Sons, Ltd.     

Multivariate analysis of combined Raman and fibre‐optic reflectance spectra for the identification of binder materials in simulated medieval paints

The non‐invasive identification of paint materials used in works of art is essential, both for preserving and restoring them, and also for understanding and verifying the history surrounding their creation. As such, the development of suitable non‐invasive techniques has received much interest in recent years. We have investigated the use of Fourier transform (FT)‐Raman spectroscopy and fibre‐optic reflectance spectroscopy (FORS), together with multivariate principal‐component analysis (PCA) techniques, in order to identify the pigment and binding materials used in made‐up samples representative of real artwork. We demonstrate that both types of spectroscopy provide complementary information which can be used to identify the pigments and binders in paint samples. We show that PCA with FT‐Raman spectra can be used to assist in the identification of oil‐based binders, and that the additional data provided by FORS spectra enables PCA on combined spectra to identify more complex proteinaceious and polysaccharide‐based binding media. The results presented here demonstrate that multivariate analyses of lead‐based paints, using data measured by FT‐Raman and FORS in conjunction, have much potential for identifying individual pigments and binders in paint samples. This provides a path towards computer‐assisted characterisation of paint materials on artwork. Copyright © 2013 John Wiley & Sons, Ltd.     

Raman microscopy of prehistoric paintings in French megalithic monuments

Remains of pictorial decorations in a series of six representative megalithic monuments of Brittany (France) and two French stelae have been studied by micro‐Raman spectroscopy for the first time. Fungal colonies on the painted orthostats made it difficult to obtain in situ Raman spectra of the paint components. Nevertheless, paint micro‐specimens studied in the laboratory by micro‐Raman spectroscopy, X‐ray photoelectron spectroscopy and scanning electronic microscopy combined with energy dispersive X‐ray spectroscopy have made possible to characterise the materials present. The minerals α‐quartz, albite, microcline, muscovite, phlogopite, celadonite, beryl and anatase have been identified in the granitic rocks supporting the paintings, while dolomite and calcite are dominant in the calcareous rocky substrata. Haematite is the main component of the red pictographs, whereas amorphous carbon and manganese oxides/oxihydroxides have been used in the black ones. Calcite, gypsum and amorphous carbon have been detected as additional components of the paint in some cases. Contamination with modern tracing materials (polystyrene and ε‐copper‐phthalocyanine blue) has been detected in several cases. The presence of pigments as decorative elements in megalithic monuments of Western France and its possible relation with those of the Iberian Peninsula create interesting expectations for the knowledge of the European megalithic culture. Copyright © 2015 John Wiley & Sons, Ltd.     

Drop‐coating deposition Raman spectroscopy of liposomes

Drop‐coating deposition Raman (DCDR) spectroscopy was tested as a potential technique for studying liposomes at very low sample concentrations. We used model liposomes prepared either from 1,2‐distearoyl‐sn‐glycero‐3‐phospocholine or from soybean asolectin, which is composed of various lipids and thus represents a good model of natural membranes. In both cases, deposited samples formed a dried drop with a circular shape with a ring of concentrated liposomes at the edge. Spectral mapping showed that maximum Raman intensity originated from the inner part of the edge ring, while Raman signal gradually decreased in both radial directions. The Raman spectra exhibited excellent reproducibility of spectral characteristics at different locations in the drop, indicating similar conformation and ordering of hydrocarbon lipid chains in the sample. Our results suggest that DCDR spectroscopy can be used for studying lipids in situ, and sensitivity of this technique is at least two orders of magnitude higher than that of conventional Raman microscopy. Copyright © 2011 John Wiley & Sons, Ltd.     

Identification of reddish pigments in octocorals by Raman spectroscopy

The nature of the pigments in octocorals has been investigated by Raman spectroscopy, where laser excitation at 632.8 and 1064 nm were used to characterize the colored components present in the skeleton of the exotic pink‐yellow soft coral Chromonephthea braziliensis, the reddish purple sea fan Leptogorgia punicea and the endemic deep violet red Leptogorgia violacea from the southeastern coast of Brazil. The observed positions of two major Raman bands at ca 1500 cm⁻¹ [ν(CC)] and 1130 [ν(C C)] for all specimens strongly suggest the presence of a mixtureof conjugated polyenes belonging to a class of compounds named parrodienes. The hemiketal steroidal feeding deterrent, 23‐keto‐cladiellin‐A, isolated from C. braziliensis was identified in the crude extracts by the Raman analysis using 1064 nm excitation. The observation of the most important vibrational bands of this compound can be useful in future investigations to monitor its presence in crude extracts of C. braziliensis and or other species. Copyright © 2010 John Wiley & Sons, Ltd.     

2D Raman spectroscopy study of dolomite and cyanobacterial extracellular polymeric substances from Khor Al‐Adaid sabkha (Qatar)

Dolomite precipitation, limited at low temperature, appears to be impacted by microbial extracellular polymeric substances (EPS). The presence of dolomites has been reported in the extreme environments of Arabian Gulf sabkhas. Many of these sites are characterized by extensive growth of cyanobacterial mats that are recognized as key producers of EPS. However, no information has been gathered on the cyanobacterial EPS involvement in dolomite precipitation. The objective of this study was to obtain in situ information on the spatial distribution of cyanobacterial EPS and dolomite in Khor Al‐Adaid sabkha (Qatar) sediments by chemical mapping. For this purpose, in situ 2D Raman spectroscopy and atomic force microscopy were applied. Additionally, samples were analyzed with scanning electron microscopy and X‐ray diffraction. Raman fingerprints of dolomite (300, 725, and 1098 cm⁻¹), cyanobacteria, and their EPS (1000, 1130, 1148, and 1508 cm⁻¹) were observed widely distributed in the top 2 cm of the sabkhas sediments. 2D chemical imaging of sediment layers characterized minerals and organic matter of microbial origins at high spatial resolution. Raman mapping indicated small dolomite clusters (<2 µm) embedded in a dense cyanobacterial EPS matrix. The spatial distribution showed that small dolomite clusters are closely associated with cyanobacterial EPS and organic carbon. Our results prove that cyanobacterial molecules are closely related to dolomite in the sabkhas sediments. This study demonstrated that Raman mapping is a robust and sensitive technique for acquisition of in situ information on cell–mineral interactions. Copyright © 2013 John Wiley & Sons, Ltd.     

In situ monitoring of solid‐state transition of p‐aminobenzoic acid polymorphs using Raman spectroscopy

The purpose of this study is to investigate the mechanism of solid‐state polymorphic transition of p‐aminobenzoic acid (PABA) using in situ Raman spectroscopy measurement. The polymorphic transition experiments were conducted on a micro quartz vessel mounted on a microscope, hot and cold stage, under isothermal conditions. The temperature was precisely controlled by a standalone temperature controller equipped with liquid nitrogen cooling system. The Raman spectroscopy probe was positioned on the surface of the solid sample in the micro vessel. The polymorphic transition progression was in situ monitored and recorded by Raman spectroscopy. Based on the polymorphic transition rate resulted from the quantitative analysis of Raman spectra, the mechanism of solid‐state polymorphic transition of PABA was examined by various empirical kinetic models. An Arrhenius analysis was also performed to calculate activation energies from 134.7 kJ mol⁻¹ to 137.7 kJ mol⁻¹ for the transition. The results demonstrated that in situ Raman spectroscopy is a valuable and accurate technique to probe polymorphic transition process. Copyright © 2009 John Wiley & Sons, Ltd.     

In situ monitoring of the solution‐mediated polymorphic transformation of glycine: characterization of the polymorphs and observation of the transformation rate using Raman spectroscopy and microscopy

The aim of this work was to investigate the mechanism and progression of the solution‐mediated polymorphic transformation and crystallization of glycine. The identification of the α‐ and γ‐forms of glycine crystals was performed using powder X‐ray diffraction (PXRD), Raman microscopy and in situ probe Raman spectroscopy. The influence of the addition of NaCl and of the process parameters such as saturation temperature, seed size and stirring speed on the transformation behavior from the metastable α‐ form to the stable γ‐ form was examined. In situ probe Raman spectroscopy was used to monitor the solid‐phase properties—polymorphic composition. Fourier transform infrared spectroscopy (FTIR) with a ZnSe window was used to track the liquid‐phase concentration at different times. Besides, the polymorphic transformation of glycine in the solvent was also examined in situ using a microscope with a heating/cooling stage. The integration of the different offline and in situ analytical measurement techniques greatly assisted in accurately and quantitatively perceiving the fundamental phenomena that govern the transformation process. Copyright © 2008 John Wiley & Sons, Ltd.     

Some forensic applications of a combined micro‐Raman and scanning electron microscopy system

The structural chemical analyser (SCA) is a novel accessory that allows the analytical advantages of Raman spectroscopy and scanning electron microscopy with energy dispersive x‐ray detection (SEM/EDX) to be realised in a single hybridised instrument. The combined Raman–SEM/EDX system permits in situ characterisation of a sample based on both its molecular and elemental makeup. This article demonstrates the potential of using the SCA for interrogating trace evidence for criminalistic purposes. Illustrative evidentiary examples (taken from our laboratory's archives) include the examination of a white paint fragment consisting of several layers of the same colour and a sample of explosive mixture recovered from a place of interest. The sensitive SEM imaging contrast mechanisms enabled the optically identical multiple layers of the white paint to be distinguished easily. The individual layers were then unambiguously analysed to establish their elemental profile (from energy dispersive x‐ray (EDX)) and this was cross‐referenced with the chemical information derived from in situ Raman measurements. X‐ray mapping was used as a fast and convenient way of characterising simultaneously multiple solids constituting the explosive mixture. Typical particles were targeted and analysed both by EDX and Raman spectroscopy revealing an unusual chlorate‐based energetic mixture that also contained 2, 4, 6‐trinitrotoluene (TNT) and 2, 4, 6‐trinitrophenylmethylnitramine (Tetryl). Copyright © 2009 John Wiley & Sons, Ltd.     

Tobacco alkaloids analyzed by Raman spectroscopy and DFT calculations

The tobacco alkaloids: nicotine, nornicotine, cotinine, anabasine, their protonated forms, and salts were analyzed by means of Raman spectroscopy supported by the density functional theory/B3LYP/aug‐cc‐pVDZ calculations. The analyses were performed based on Raman marker bands of neutral, monoprotonated, and diprotonated forms of tobacco alkaloids because in different surroundings various forms have been suggested to either dominate or to coexist. The form and distribution of nicotine directly in a plant and in phytopharmaceutical products were investigated by in situ Raman mapping. For the first time, the Raman optical activity spectrum of (−)‐nicotine in aqueous solution was measured and interpreted by means of the density functional theory calculations. The study provides a clear evidence that Raman spectroscopy techniques are powerful in efficient quality control and forensic and bioanalytical analyses of tobacco alkaloids. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of epidermal L‐tryptophan and its oxidation products by in vivo FT‐Raman Spectroscopy further supports oxidative stress in patients with vitiligo

In the past, non‐invasive in vivo FT‐Raman spectroscopy has been used to detect H₂O₂‐mediated oxidation of methionine to methionine sulfoxide and methionine sulfone, as well as cysteine to cysteic acid, in the sequence of proteins in the epidermis of patients with vitiligo. L ‐tryptophan (Trp) is another potential target for this oxidation. Owing to the presence of 10⁻³M epidermal albumin which contains one Trp residue, it was tempting to follow the oxidation of this amino acid. Using in vivo and in vitro FT‐Raman spectroscopy, we show for the first time that epidermal Trp is oxidised in patients with vitiligo, yielding 5‐OH‐Trp at 930 cm⁻¹ and other oxidation products (i.e. N‐formyl kynurenine and kynurenine) from indole ring oxidation peaking at 1050 cm⁻¹. On the basis of detailed in vitro results, we could conclude that 5‐OH‐Trp as well as formyl kynurenine and kynurenine are formed via H₂O₂‐mediated Fenton chemistry. These results once again bring out the strength of non‐invasive in vivo FT‐Raman Spectroscopy in dermatology to follow the effect of oxidative stress in the skin of patients with vitiligo. Copyright © 2008 John Wiley & Sons, Ltd.     

Colour diversification in octocorals based on conjugated polyenes: A Raman spectroscopic view

Polyenic pigments in octocorals have been investigated by Raman spectroscopy using laser excitation at 532, 785 and 1064 nm. The spectral features suggest the structural nature of carotenoids from Phyllogorgia dilatata, Leptogorgia punicea, Muricea atlantica, Carijoa riisei and conjugated polyenals from L. punicea, L. setacea, Muricea flamma and Renilla muelleri. The observed vibrational bands at ca. 1540–1520 ν₁(C=C), 1159 ν₂(C–C) and 1005 cm⁻¹ ρ₃(C–CH₃) were assigned to carotenoids, whereas the identification of non‐methylated conjugated polyenals have been proposed due to two major Raman bands at ca. 1500 and 1120 cm^‐1, assigned to ν(C=C) and ν(C–C), respectively. Copyright © 2012 John Wiley & Sons, Ltd.     

In‐situ and laboratory Raman analysis in the field of cultural heritage: the case of a mural painting

The present work exemplifies, over a mural painting from the 14th century, the advantages of an initial exhaustive research using latest generation hand‐held spectrometers (Raman mainly) in order to perform the characterization of valuable objects of cultural heritage. These in‐situ techniques (meaning on‐site and non‐destructive) are very useful to study the pigments and materials, to identify the nature and causes of some of the main sources of deterioration and to examine past repaints. In addition, the in‐situ measurements are of great importance in the selection of micro‐samples for the laboratory analyses. In this particular case, the combination of these results with the chemical imaging analyses in the laboratory (such as Raman and energy dispersive X‐ray spectrometry imaging) allowed the characterization of the mural painting, including, the identification of all restoration works applied in the past. Copyright © 2014 John Wiley & Sons, Ltd.     

Modeling red coral (Corallium rubrum) and African snail (Helixia aspersa) shell pigments: Raman spectroscopy versus DFT studies

Pigments from red coral (Corallium rubrum) and African snail (Helixia aspersa) shell were studied non‐invasively using Raman spectroscopy with 1064‐nm laser beam. The two observed bands because of organic pigments confined in biomineralized CaCO₃ matrix at about 1500 and 1100 cm⁻¹ were assigned to ν(CC) and ν(C―C), respectively. Both signals originate from polyene(s) of largely unknown structure, containing several conjugated CC bonds. The small peak at 1016 cm⁻¹ in the Raman spectrum of coral pigment was assigned to in‐plane ―CH₃ rocking or structural deformation of polyene chain because of spatial confinement in the mineral matrix. The organic pigments in red coral and snail shell were present in inorganic matrix containing aragonite (shell) and calcite (coral). In addition, using Raman spectroscopy, it was observed that aragonite was replaced by calcite as result of healing damaged parts of snail shell. This is an important finding which indicates a great potential of nondestructive Raman spectroscopy instead of X‐ray technique, as a diagnostic tool in environmental studies. To support analysis of the observed Raman spectra detailed calculations using density functional theory (DFT with B3LYP and BLYP density functionals) on structure and vibrations of model all‐trans polyenes were undertaken. DFT calculated CC and C―C stretching frequencies for all‐trans polyenes containing from 2 to 14 CC units were compared with the observed ν(CC) and ν(C―C) band positions of the studied coral and shell. Individual correction factors were used to better match theoretical wavenumbers with observed band positions in red coral and African snail. It was concluded that all‐trans polyene pigments of red coral and dark parts of African snail shell contain 11–12 and 14 CC double bond units, respectively. However, Raman spectroscopy cannot produce any clear information on the presence and nature of the end‐chain substituents in the studied pigments. Copyright © 2016 John Wiley & Sons, Ltd.