Investigation of crimson-dyed fibres for a new approach on the characterization of cochineal and kermes dyes in historical textiles

The colorant behaviour of cochineal and kermes insect dyes in 141 experimentally-dyed and 28 artificially-aged samples of silk and wool was investigated using ultra-high performance liquid chromatography coupled to photodiode array detector (UHPLC-PDA), liquid chromatography electrospray ionisation mass spectrometry (LC-ESI-MS) and image scanning electron microscopy – energy dispersive X-ray spectroscopy (SEM-EDX). Partial-least squares discriminant analysis (PLS-DA) was then used to model the acquired UHPLC-PDA data and assess the possibility of discriminating cochineal insect species, as well as their correspondent dyed and aged reference fibres. The resulting models helped to characterize a set of 117 red samples from 95 historical textiles, in which UHPLC-PDA analyses have reported the presence of cochineal and kermes insect dyes.     

Structure elucidation and chromatographic identification of anthraquinone components of cochineal (Dactylopius coccus) detected in historical objects

Cochineal is one of the most well known organic red dyes. Dactylopius coccus Costa (Dactylopiidae) is a scale insect that is used as the source of the dye known as Mexican cochineal. Although cochineal is today a natural food colorant (E120) and although it has been used in art objects (textiles and paintings) for centuries, its exact chemical consistency is not well clarified except for carminic acid which is the major component and kermesic and flavokermesic acids. Several minor components (typically less than 5% of the colouring material) remained unknown or partially studied, although their presence has been reported in numerous analytical works related to art objects. Chemical investigation of the methanol extract of the dried insects, after subsequent HPLC chromatographic separations, led to the isolation and structure elucidation of six new anthraquinones, along with the known compounds carminic acid, kermesic acid and flavokermesic acid. The new compounds formerly described as DCII and DCIII, were found to be the 2-C-glucoside of flavokermesic acid and 4-aminocarminic acid, respectively, while DCIV and DCVII were found to be the α/β C-glucofuranosides of kermesic acid, and were studied as a mixture due to equilibrium. In addition, 3-O-glucoside of flavokermesic acid (DCOFK), and 3,4-dideoxycarminic acid (DDCA) were identified. The structures of the new compounds were elucidated on the basis of their NMR and MS data. Finally, the new compounds were detected in silk dyed with cochineal, lake pigment and, furthermore, in historical objects of the cultural heritage (icon and textile) using LC–DAD and LC–MS.     

Flavylium chromophores as species markers for dragon's blood resins from Dracaena and Daemonorops trees

A simple and rapid liquid chromatographic method with diode-array UV-vis spectrophotometric detection has been developed for the authentication of dragon's blood resins from Dracaena and Daemonorops trees. Using this method it was discovered that the flavylium chromophores, which contribute to the red colour of these resins, differ among the species and could be used as markers to differentiate among species. A study of parameters, such as time of extraction, proportion of MeOH and pH, was undertaken to optimise the extraction of the flavyliums. This method was then used to make extracts from samples of dragon's blood resin obtained from material of known provenance. From the samples analysed 7,6-dihydroxy-5-methoxyflavylium (dracorhodin), 7,4'-dihydroxy-5-methoxyflavylium (dracoflavylium) and 7,4'-dihydroxyflavylium were selected as species markers for Daemonorops spp., Dracaena draco and Dracaena cinnabari, respectively. The chromatograms from these samples were used to build an HPLC-DAD database. The ability to discriminate among species of dragon's blood using the single marker compounds was compared with a principal components analysis of the chromatograms in the HPLC-DAD database. The results from the HPLC-DAD method based on the presence of these flavylium markers was unequivocal. The HPLC-DAD method was subsequently applied to 37 samples of dragon blood resins from the historical samples in the Economic Botany Collection, Royal Botanic Gardens, Kew. The method identified anomalies in how samples in this collection had been labelled. It is clear that the method can be used to evaluate the provenance of samples used in different areas of cultural heritage. It also could be used to monitor the trade of endangered species of dragon's blood and the species being used in complex formulations of traditional Chinese medicine.     

The natural constituents of historical textile dyes

The sources and structures of dyes used to colour Western historical textiles are described in this tutorial review. Most blue and purple colours were derived from indigo--obtained either from woad or from the indigo plant--though some other sources (e.g. shellfish and lichens) were used. Reds were often anthraquinone derivatives obtained from plants or insects. Yellows were almost always flavonoid derivatives obtained from a variety of plant species. Most other colours were produced by over-dyeing--e.g. greens were obtained by over-dyeing a blue with a yellow dye. Direct analysis of dyes isolated from artefacts allows comparison with the historical record.     

Novel methodology for the extraction and identification of natural dyestuffs in historical textiles by HPLC-UV-Vis-ESI MS. Case study: chasubles from the Wawel Cathedral collection

High-performance liquid chromatography coupled with spectrophotometric and electrospray mass spectrometric detection (HPLC-UV-Vis-ESI MS) was used for characterization of natural dyes present in historical art works. The gradient program was developed for identification of 29 colorants of various polarities. Dual detection system (UV-Vis and ESI MS) allowed differentiation of all compounds, even if they were not completely separated. This enabled examination of more color compounds over a substantially shorter time in comparison with previously recommended methods. Moreover, for extraction of colorants from historical textiles a two-step sequential procedure was proposed, excluding evaporation used in earlier procedures. The developed method was successfully applied to identification of indigotin, carminic, kermesic, flavokermesic, dcII, dcIV, dcVII, and ellagic acids as well as luteolin, apigenin, and genistein in red, violet, and green fibers taken from three selected historical chasubles which belong to the collection of the Wawel Cathedral treasury (Cracow, Poland). Italian textiles from the fifteenth and sixteenth centuries, of which chasubles were made, were dyed with a limited number of dyestuffs, consistently used for all batches of fabrics. The obtained results also allowed confirmation of the structure of the so-called "dcII" component of cochineal as a C-glucose derivative of flavokermesic acid.     

Extracting natural dyes from wool--an evaluation of extraction methods

The efficiency of eight different procedures used for the extraction of natural dyes was evaluated using contemporary wool samples dyed with cochineal, madder, woad, weld, brazilwood and logwood. Comparison was made based on the LC-DAD peak areas of the natural dye's main components which had been extracted from the wool samples. Among the tested methods, an extraction procedure with Na(2)EDTA in water/DMF (1:1, v/v) proved to be the most suitable for the extraction of the studied dyes, which presented a wide range of chemical structures. The identification of the natural dyes used in the making of an eighteenth century Arraiolos carpet was possible using the Na(2)EDTA/DMF extraction of the wool embroidery samples and an LC-DAD-MS methodology. The effectiveness of the Na(2)EDTA/DMF extraction method was particularly observed in the extraction of weld dye components. Nine flavone derivatives previously identified in weld extracts could be identified in a single historical sample, confirming the use of this natural dye in the making of Arraiolos carpets. Indigo and brazilwood were also identified in the samples, and despite the fact that these natural dyes were referred in the historical recipes of Arraiolos dyeing, it is the first time that the use of brazilwood is confirmed. Mordant analysis by ICP-MS identified the widespread use of alum in the dyeing process, but in some samples with darker hues, high amounts of iron were found instead.     

Electrochemical analysis of natural solid organic dyes and pigments

Square-wave voltammetry of solid naphthoquinone, anthraquinone, and flavone dyes, carmine, cochineal red, indigo, and Prussian blue, was compared to microanalysis (sample consumption <1 mg) of traditional painting pigments and dyes without their preliminary dissolution. Electrochemical analysis was also performed after the samples' hydrolysis simultaneously with thin-layer chromatography. Anthraquinone-based pigments and Prussian blue are reversibly reduced, cochineal red and lac dyes are irreversibly reduced, flavones are mostly reversibly oxidized, dragon's blood is irreversibly oxidized and reduced, and indigo yields both reversible oxidation and reduction. The potential window of these reactions is about 1.4 V wide. This variability permits identification of the kind of pigment or dye, and directly distinguishes, for example, alizarin and purpurin; luteolin and quercetin; or indigo and Prussian blue.     

A new chromatographic method for the separation of food dye mixtures on thin MgO layers

Summary A new effective TLC method has been developed for the separation of the five main watersoluble food dyes such as indige carmin, cochineal red, acid amaranth I, orange yellow S and tartrazine G. The chromatograms are developed by the ascending technique on thin MgO layers applied onto glass plates, previously activated at 130°C. A 82 mixture of 15% sodium citrate and methanol is used as the developing phase. This composition was selected on the basis of Snyder's theory and the polarity index which for this mixture is 8.3. The proposed method permits the full separation of the dye mixture from a 0.3l sample solution with concentrations of 1×10^–6 mole/liter.     

Liquid chromatography determination of natural dyes in extracts from historical Scottish textiles excavated from peat bogs

Textiles excavated from Scottish sites belonging now to the collections of the National Museums of Scotland, including seventeenth century textiles from peat bogs in the Scottish Highlands and Islands, were selected for analysis by high performance liquid chromatography with photodiode array detection (PDA HPLC) to detect whether any dyes remained and, if so, to identify their biological sources. Dye components were identified in 36 of the 81 samples analysed. Although it was not possible to identify the exact sources of the dyestuffs because of the wide-spread occurrence of these natural dyes components, the study has shown that textiles previously not thought to have been coloured had detectable traces of dye. Before the historical textiles were analyzed, an improved extraction procedure that combined the routine acid hydrolysis method with one using dimethylformamide (DMF) was applied. The DMF method enabled increased recovery of major flavonoid and anthraquinoid compounds, and very high efficiency of recovery of indigotin even in textiles with no colour visible, thereby complementing the acid hydrolysis method already in use. Extracts from historical thread samples were analysed by PDA HPLC using a reversed-phase gradient system comprising of a C18 column (150 mm x 4.6 mm i.d., 25 +/- 1 degrees C) with water, methanol and o-phosphoric acid at an eluent flow rate of 1.2 ml/min. A preliminary investigation to improve the detection limits further for a selection of natural dyes was made by comparing results from the 4.6mm internal diameter (i.d.) column with a narrow bore C18 column (2.1 mm i.d.). An increase in the detector response was observed for narrow-bore column proving its possibility of enhancement of sensitivity.     

Identification of naphthoquinonic and anthraquinonic dyes via sequential potential steps applied to the voltammetry of microparticles methodology

An electrochemical method for identifying anthraquinonic, naphthoquinonic, and related dyes in microsamples is reported. This method is based on the sequential application of oxidative and reductive constant-potential polarization steps coupled with the record of square wave voltammograms to solid microsamples of dyes in contact with aqueous electrolytes. As a result, oxidized/reduced products form a layer on the lateral faces of the dye crystals as suggested by attenuated total reflectance-Fourier transform infrared spectroscopy and atomic force microscopy data. This methodology is applied for characterizing alizarin, purpurin, and natural dyes aloe, cochineal red, madder lake, kermes, shellac, and henna attached to paraffin-impregnated graphite electrodes in contact with aqueous potassium phosphate buffer.     

Identification of anthraquinone coloring matters in natural red dyestuffs by high performance liquid chromatography with ultraviolet and electrospray mass spectrometric detection

Reversed phase liquid chromatography with diode array detection (DAD) and electrospray mass spectrometric (ESI MSD) methods were developed for the identification of anthraquinone color components of cochineal, lac dye, and madder – red natural dyestuffs. Electrospray mass spectrometry was found to be more suitable than diode array detection for such analysis because of its higher sensitivity (detection limits in the range 30–90 ng mL^–1) and selectivity. The developed method permitted unequivocal identification of carminic acid and laccaic acid A as coloring matters in examined preparations of cochineal and lac dye, respectively. In madder more chemical color species were found: alizarin, purpurin, lucidin, ruberythric acid, and also aluminum and calcium alizarin lake. Among the methods recommended so far, the present one allows fast, direct, and unequivocal identification of components of very complicated natural products used in art.     

Investigation of the colourants used in icons of the Cretan School of iconography

The red shades of 13 icons (15th-17th century) of the Cretan School of iconography are investigated in detail to identify the inorganic and organic colouring materials comprising the paint layers. Examination of sample cross-sections is performed with optical microscopy. Micro-Raman spectroscopy and high performance liquid chromatography (HPLC) coupled to a photodiode array detector are employed for the identification of the inorganic and organic colouring materials, respectively. The results reveal the extensive use of coccid dyes by the Cretan painters: kermes (Kermes vermilio Planchon) is found in icons dated before the middle 16th century and cochineal in icons created several decades after the discovery of the New World. Other dyestuffs detected in the historical samples are madder (possibly Rubia tinctorum L., according to HPLC profiles), soluble redwood and indigoid dyes. Organic dyes were used by the painters as exclusive colouring matters (or glazes) or in mixtures with inorganic pigments, such as red ochre, cinnabar, minium, azurite lead white and carbon black.Liquid chromatography with mass spectrometry (LC-MS) coupled to a negative electrospray ionization mode is employed to provide information on the identity of some unknown colouring components, of the aforementioned dyes, detected in the historical samples. The results suggest that (i) the type B compound (also known as Bra′) is a dehydro-brazilein product and (ii) the deprotonated molecular ion of the type C compound corresponds to m/z = 243. Both compounds are commonly used as markers for the identification of soluble redwood in historical samples. LC-MS analysis of cochineal shows that the dcIV and dcVII components are isomeric with carminic acid, as it has been recently suggested. Finally, LC-MS is employed to identify and record kermesic and flavokermesic acid in kermes and rubiadin in wild madder.     

Analysis of organic colouring and binding components in colour layer of art works

Two methods of analysis of organic components of colour layers of art works have been tested: IR microspectroscopy of indigo, Cu-phthalocyanine, and Prussian blue, and MALDI-TOF-MS of proteinaceous binders and a protein-containing red dye. The IR spectra distortion common for smooth outer surfaces and polished cross sections of colour layer of art works is suppressed by reflectance measurement of microtome slices. The detection limit of the three blue pigments examined is ~0.3 wt% in reference colour layers in linseed oil binder with calcite as extender and lead white as a drying agent. The sensitivity has been sufficient to identify Prussian blue in repaints on a Gothic painting. MALDI-TOF-MS has been used to identify proteinaceous binders in two historical paintings, namely isinglass (fish glue) and rabbit glue. MALDI-TOF-MS has also been proposed for identification of an insect red dye, cochineal carmine, according to its specific protein component. The enzymatic cleavage with trypsin before MALDI-TOF-MS seems to be a very gentle and specific way of dissolution of the colour layers highly polymerised due to very long aging of old, e.g. medieval, samples.     

A precise self-built secondary mass database for identifying red dyes and dyeing techniques with UPLC-MS/MS

Dyes on ancient silks have been a worth studying field through human's history, although current reports ignore the connection between natural dyes origin and relevant colour reduction methods, which poses an insurmountable obstacle for restoration of historical silks. In this paper, a series of 12 red hue silks from six natural dyes (sappanwood, Chinese madder, safflower, lac, cochineal, dragon's blood) via three different dyeing techniques were used to establish a self-built precise tandem mass spectrometry (MS/MS) database. With organic solvent extracting on those manual-dyed silks, ultraperformance liquid chromatography - electrospray ionization - quadrupole time of flight mass spectrometry (UPLC-ESI-QTOF-MS) was utilized to form preliminary MS database for screening and identifying of the potential dyes compounds without standard references. Furthermore, combining the targeted MS/MS mode and the matching threshold of 70.00, a self-built secondary MS/MS database was successfully established, which contains 33 compounds, 32 chromatograms and 32 MS/MS fragments. As for real sample application, the self-built precise MS/MS database had revealed that the dyes on two historical silks (Shanghai Museum, China) belong to Chinese madder just with different mordant dyeing ordinal. Additionally, by experimental restoration, visually indistinguishable silks (ΔE_ab* < 1.5 NBS) were successfully restored. This explorative methodology can further inspire the traceability of biological dyestuffs, which lays instructive foundation on protection and restoration of artefacts, connecting the archaeological science and human art.     

液相色谱-二极管阵列检测/质谱联用在活性染料结构鉴定中的应用

基于多年运用液相色谱-二极管阵列检测/质谱(HPLC-DAD/MS)联用技术分析活性染料的实验积累,总结了HPLC-DAD/MS联用方法在商品活性染料结构鉴定中的重要应用。商品染料不需要纯化即可通过单次进样达到复杂组分的分离并得到光谱和质谱信息;结合衍生物质量差值法可推定染料的活性基种类和数目;采用二极管阵列检测器的全光谱扫描功能可以得到染料的发色体类别等有用的信息。HPLC-DAD/MS联用方法简便、快速、准确,尤其适用于多组分拼混的商品染料的结构鉴定,有较大的实用价值。通过对多组分商品染料活性灰、活性深黑、海洋蓝中关键组分的结构分析实例,介绍了HPLC-DAD/MS联用分析技术在未知商品活性染料结构分析中的应用。     

Microextraction of Reseda luteola-Dyed Wool and Qualitative Analysis of Its Flavones by UHPLC-UV,NMR and MS

Detailed knowledge on natural dyes is important for agronomy and quality control as well as the fastness, stability, and analysis of dyed textiles. Weld (Reseda luteola L.), which is a source of flavone-based yellow dye, is the focus of this study. One aim was to reduce the required amount of dyed textile to ≤50 μg for a successful chromatographic analysis. The second aim was to unambiguously confirm the identity of all weld flavones. By carrying out the extraction of 50 μg dyed wool with 25 μL of solvent and analysis by reversed-phase UHPLC at 345 nm, reproducible chromatographic fingerprints could be obtained with good signal to noise ratios. Ten baseline separated peaks with relative areas ≥1% were separated in 6 min. Through repeated polyamide column chromatography and prepHPLC, the compounds corresponding with the fingerprint peaks were purified from dried weld. Each was unequivocally identified, including the position and configuration of attached sugars, by means of 1D and 2D NMR and high-resolution MS. Apigenin-4′-O-glucoside and luteolin-4′-O-glucoside were additionally identified as two trace flavones co-eluting with other flavone glucosides, the former for the first time in weld. The microextraction might be extended to other used dye plants, thus reducing the required amount of precious historical textiles.     

Peak purity determination with principal component analysis of high-performance liquid chromatography-diode array detection data

A method is proposed for the determination of chromatographic peak purity by means of principal component analysis (PCA) of high-performance liquid chromatography with diode array detection (HPLC-DAD) data. The method is exemplified with analysis of binary mixtures of lidocaine and prilocaine with different levels of separation. Lidocaine and prilocaine have very similar spectra and the chromatograms used had substantial peak overlap. The samples analysed contained a constant amount of lidocaine and a minor amount of prilocaine (0.02-2 conc.%) and hence the focus was on determining the purity of the lidocaine peak in the presence of much smaller levels of prilocaine. The peak purity determination was made by examination of relative observation residuals, scores and loadings from the PCA decomposition of DAD data over a chromatographic peak. As a reference method, the functions for peak purity analysis in the chromatographic data system used (Chromeleon) were applied. The PCA method showed good results at the same level as the detection limit of baseline-separated prilocaine, outperforming the methods in Chromeleon by a factor of ten. There is a discussion of the interpretation of the result, with some comparisons with evolving factor analysis (EFA). The main advantage of the PCA method for determination of peak purity over methods like EFA lies in its simplicity, short time of calculation and ease of use.     

Preparation of highly selective solid-phase extractants for Cibacron reactive dyes using molecularly imprinted polymers

Selective polymeric extractants were prepared for preconcentration of Cibacron reactive red dye, a dye that is often applied with Cibacron reactive blue and Cibacron reactive yellow for dyeing of fabrics. The best extractant was fabricated (in chloroform) using methacrylic acid (as monomer), ethylene glycol dimethacrylate (as crosslinker), AIBN (as initiator for polymerization), and red dye as template molecule, with a molar stoichiometric ratio of 8.0:40.0:2.5:0.63, respectively. The structure of the molecularly imprinted polymer (MIP) was robust, and resisted dissolution up to 260 °C. Compared with the un-imprinted polymer, the imprinted product has a large specific surface area which improved its adsorption capacity. The effect of imprinting was obvious from the adsorption capacity measured at pH 4 for red dye (the imprinted molecule), which was increased from 24.0 to 79.3 mg g⁻¹ after imprinting. Equilibrium adsorption studies revealed that the dye-imprinted-polymer enables efficient extraction of red dye even in the presence of blue and yellow dyes which have similar chemical natures to the red dye. The selectivity coefficients S _{red dye/dye}, were 13.9 and 17.1 relative to the yellow and blue dyes, respectively. The MIP was found to be effective for red dye preconcentration, with a preconcentration factor of 100, from tap water and treated textile wastewater. The factors affecting extraction of red dye by the MIP were studied and optimized. Under the optimized extraction conditions, red dye was selectively quantified in the presence of other competing dyes at a concentration of 20 μg L⁻¹ from different water systems with satisfactory recoveries (91–95%) and RSD values (∼5.0%).     

High-performance liquid chromatographic determination of colouring matters in historical garments from the Holy Mountain of Athos

This work is probably the first attempt to identify the organic colouring materials contained in post-Byzantine textiles, from the Holy Mountain of Athos. Samples extracted from seven ecclesiastical garments (15th–19th century) are investigated by high performance liquid chromatography with UV-Vis diode array detection. The detection limits for alizarin, purpurin, carminic acid, laccaic acid A, luteolin, apigenin, genistein, fisetin, sulfuretin, ellagic acid, indigotin and indirubin are found to be within 0.002–0.029 μg mL⁻¹. The following organic dyes are identified in the extracts: dyer’s broom (Genista tinctoria L.), young fustic (Cotinus coggygria Scop.), an indigoid dye source either indigo (Indigofera species) or woad (Isatis tinctoria L.), madder, cochineal and lac dye (Kerria lacca Kerr). Furthermore, the identification of a brazilein derivative indicates the presence of a Caesalpinia dye source in the samples. Correspondence: Ioannis Karapanagiotis, Ormylia Art Diagnosis Center, Sacred Convent of the Annunciation, Ormylia, GR-63071 Chalkidiki, Greece     

Microwave-assisted rapid extraction of red dye from Caesalpinia sappan heartwood

Since ancient times Caesalpinia sappan heartwood dye has been well-known for its medicinal and dyeing properties. Isolation of the red dye using both conventional and newly developed microwave method was carried out. The conventional heating of 2 h provided 0.656 +/- 0.049 g of the dye and by microwave heating at 540 W for 20 min, the yield obtained was 0.747 +/- 0.047 g. Both the dyes were found to be the same as evidenced by UV, TLC and HPTLC studies. Antioxidant activity of the dyes was also carried out using DPPH and nitric oxide methods to confirm the similarity in their biological activity. The procedure developed can be used for the fast extraction of the red dye of C. sappan without affecting the nature of the product.