Identification of anthraquinone coloring matters in natural red dyes by electrospray mass spectrometry coupled to capillary electrophoresis

Capillary electrophoresis with UV/visible diode-array detection (DAD) and electrospray mass spectrometric (ESI-MS) detection were used for the identification of anthraquinone color components of cochineal, lac-dye and madder, natural red dyestuffs often used by ancient painters. For the purpose of such analysis, ESI-MS was found to be a much more appropriate detection technique than DAD one owing to its higher sensitivity (detection limits in the range 0.1-0.5 micro g ml(-1)) and selectivity. The method developed made it possible to identify unequivocally carminic acid and laccaic acids A, B and E as coloring matters in the examined preparations of cochineal and lac-dye, respectively. In madder, European Rubia tinctorum, alizarin and purpurin were found. The method allows the rapid, direct and straightforward identification and quantification of components of natural products used in art and could be very helpful in restoration and conservation procedures.     

Investigation of red natural dyes used in historical objects by HPLC-DAD-MS

High performance liquid chromatography (HPLC) with UV-Vis Diode Array Detection (DAD) and electrospray mass spectrometric (ESI-MS) method was utilized for the identification of coloring components of madder, Armenian and Mexican cochineal, lac dye, brazilwood, safflower and dragon blood--probably the most important red natural dyestuffs found in objects of the cultural heritage. UV-Vis detection limits in the range of 0.2-0.6 ng for carminic acid, alizarin and purpurin were achieved using a gradient elution of H2O-0.01% TFA and CH3CN-0.01% TFA. ESI mass spectrometer was also used, as a supportive detection method to the standard DAD, for further analysis of the tested materials, with the ability to analyze dyestuffs as small as one milligram. The presence of madder was revealed in two historical (Hellenistic and Roman period) samples, found in the Mediterranean area, by identifying purpurin in both of them. Munjistin was also identified in one of the samples (Hellenistic period) while alizarin was not detected, raising questions regarding the exact madder type, utilized in the historical samples.     

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     

Analysis of natural red dyes (cochineal) in textiles of historical importance using HPLC and multivariate data analysis

A new analytical approach based on high-performance liquid chromatography with diode array detector (HPLC-DAD) and multivariate data analysis was applied and assessed for analyzing the red dye extracted from cochineal insects, used in precious historical textiles. The most widely used method of analysis involves quantification of specific minor compounds (markers), using HPLC-DAD. However, variation in the cochineal markers concentration, use of aggressive dye extraction methods and poor resolution of HPLC chromatograms can compromise the identification of the precise insect species used in the textiles. In this study, a soft extraction method combined with a new dye recovery treatment was developed, capable of yielding HPLC chromatograms with good resolution, for the first time, for historical cochineal-dyed textiles. After principal components analysis (PCA) and mass spectrometry (MS), it was possible to identify the cochineal species used in these textiles, in contrast to the accepted method of analysis. In order to compare both methodologies, 7 cochineal species and 63 historical cochineal insect specimens were analyzed using the two approaches, and then compared with the results for 15 historical textiles in order to assess their applicability to real complex samples. The methodology developed here was shown to provide more accurate and consistent information than the traditional method. Almost all of the historical textiles were dyed with Porphyrophora sp. insects. These results emphasize the importance of adopting the proposed methodology for future research on cochineal (and related red dyes). Mild extraction methods and HPLC-DAD/MSⁿ analysis yield distinctive profiles, which, in combination with a PCA reference database, are a powerful tool for identifying red insect dyes.     

Micellar electrokinetic chromatography method for the determination of several natural red dyestuff and lake pigments used in art work

The identification of organic colorants used in artistic paintings is an important information source for reconstructing the working techniques found in a particular work and for defining a programme for the restoration and conservation of the painting. In this work, sodium dodecyl sulfate (SDS) was used as a surfactant in micellar electrokinetic chromatography (MEKC) for separating a broad range of red organic pigments, based on their colouring matters: madder (colouring matters: alizarin, quinizarin and purpurin), cochineal (colouring matter: carminic acid), red sandalwood (colouring matter: santalin), brazilwood (colouring matter: brazilin), lac dye (colouring matter: laccaic acid) and dragon's blood (colouring matter: dracorhodin). The running electrolyte used was 20 mM borax (pH 9), containing 20 mM SDS and 10% acetonitrile as organic modifier. Separation was carried out by applying a +20 kV voltage at the injection end, 25 degrees C and 214 nm/254 nm as detection wavelengths. All colorants were separated within less than 13 min with a good baseline resolution. The method was applied to the analysis of paint samples obtained from the Diocesan Museum of Holy Art of Bilbao.     

Identification of Natural and Early Synthetic Textile Dyes with HPLC and UV/Vis-Spectroscopy by Diode Array Detection

Abstract

The separation and identification of complex mixtures of natural and synthetic textile dyes was investigated using HPLC with diode array detection. Separation was carried out on a reversed phase column with acetonitrile-phosphoric acid gradient elution. The results show that the anthraquinones from madder root and the insect dye cocheneal present in ancient red dyes can easily be distinguished from azo-dyes present in later textile fibres. They further show that for an analysis of the numerous flavones and flavonoles, constituting most of the yellow natural dyestuffs, the combination of HPLC with on-line optical spectroscopy is particularly useful. Even when retention times are identical - as for instance for quercetin and luteolin - the ratio of a mixture can still be estimated by evaluating the ratio-chromatogram.     

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.     

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.     

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.     

High-performance liquid chromatographic determination of some anthraquinone and naphthoquinone dyes occurring in historical textiles

A reversed-phase HPLC method has been developed for identification and quantitation of nine natural quinone dyes and applied to historical textile fibres. A Purospher RP18e column was used with a convex gradient of methanol in a mobile phase of 0.1 M aqueous citrate buffer (pH 2.5) and spectrophotometric diode-array detection at 270 nm. For identification of alizarin, purpurin and xanthopurpurin, occurring together in the madder plant, an isocratic method was used with a methanol-0.2 M acetate buffer (pH 4.3) (75:25) as the mobile phase. After an acid extraction of textile fibres and the analysis of the extracts, alizarin and purpurin were identified and quantitated in three fibres.     

Electrochemical identification of anthraquinone-based dyes in solid microsamples by square wave voltammetry using graphite/polyester composite electrodes.

An electrochemical method for identifying anthraquinone-type dyes in microsamples from works of art, based in the voltammetry of microparticles approach, is reported. Upon attachment onto graphite/polyester composite electrodes, natural pigments aloe, henna, cochineal red, madder lake, kermes, shellac, and alizarin and purpurin taken as reference materials can be identified from their square wave voltammetric profiles in MeCN (0.10 mol L(-1) Bu(4)NPF(6)) and aqueous (0.25 mol L(-1) acetic acid+0.25 mol L(-1) sodium acetate) electrolytes.     

Comparison of LC–UV, LC–ELSD and LC–MS Methods for the Determination of Sesquiterpenoids in Various Species of Artemisia

Simple and specific analytical methods for the quantitative determination of sesquiterpenoids from various species of Artemisia plant samples were developed. By LC–UV, LC–ELSD, the separation was achieved by reversed-phase chromatography on a C18 column with water and acetonitrile both containing 0.025% trifluoroacetic acid as the mobile phase. In the LC–MS system, trifluoroacetic acid was replaced by 0.1% formic acid. The wavelength used for quantification of sesquiterpenoids with a diode array detector was 205 nm. The limits of detection by LC–MS was found to be 5, 10, 25, 50, 50 ng mL^?1. The limits of detection by LC–UV and LC–ELSD were found to be 5.0, 3.0, 100, 100, 7.5 μg mL^?1, by LC–UV and 50, 25, 30, 100 and 75 μg mL^?1 by LC–ELSD. LC–mass spectrometry coupled with electrospray ionization (ESI) interface is described for the identification and quantification of sesquiterpenoids in various plant samples. This method involved the use of the [M + H]⁺ ions of sesquiterpenoids in the positive ion mode with extractive ion monitoring.     

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.     

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.     

Identification of natural dyes used in works of art by pyrolysis–gas chromatography/mass spectrometry combined with in situ trimethylsilylation

Samples of four natural dyes from different organic families—natural madder (anthraquinonoid), curcuma (curcuminoid), saffron (carotenoid) and indigo (indigotic)—were analysed using a new procedure based on pyrolysis–gas chromatography/mass spectrometry (Py–GC/MS), which includes the on-line derivatisation of the natural dyes using hexamethyldisilazane (HMDS). In addition, a previous procedure involving the addition of a 10% H₂SO₄ aqueous solution to the dye and further separation with ethyl acetate has been tested. This procedure enhances the sensitivity of the method by extracting the colouring compounds from the rest of the compounds present in the natural dye. Two possible derivatising reagents—HMDS and tetramethylammonium hydroxide (TMAH)—were compared in order to assess their effectiveness in the proposed method. Characteristic peaks from trimethylsilyl derivatives of alizarin, quinizarin, xanthopurpurin and purpurin were obtained for madder; peaks from safranal, isophorone and trimethylsilyl derivative of crocetin for saffron; peaks from 4-(4-hydroxy-3-methoxy)phenyl-3-buten-2-one and 4-(4-hydroxy-3-methoxy)phenyl-2-butanone, which are primary pyrolysis products of curcuma, and peaks from indole, 2-methylindole and 2,3-dihydroindol-2-one, which are primary pyrolysis products of indigo, among others, were obtained. The reported procedure leads to the unambiguous identification of the four studied dyes from solid samples formed by individual dyes.Electronic Supplementary Material Supplementary material is available for this article at     

Simultaneous determination of eight major steroids from Polyporus umbellatus by high‐performance liquid chromatography coupled with mass spectrometry detections

Polyporus umbellatus is a widely used diuretic herbal medicine. In this study, a high‐performance liquid chromatography coupled with atmospheric pressure chemical ionization–mass spectrometric detection (HPLC‐APCI‐MS) method was developed for qualitative and quantitative analysis of steroids, as well as for the quality control of Polyporus umbellatus. The selectivity, reproducibility and sensitivity were compared with HPLC with photodiode array detection and evaporative light scattering detection (ELSD). Selective ion monitoring in positive mode was used for qualitative and quantitative analysis of eight major components and β‐ecdysterone was used as the internal standard. Limits of detection and quantification fell in the ranges 7–21 and 18–63 ng/mL for the eight analytes with an injection of 10 µL samples, and all calibration curves showed good linear regression (r² > 0.9919) within the test range. The quantitative results demonstrated that samples from different localities showed different qualities. Advantages, in comparison with conventional HPLC–diode array detection and HPLC‐ELSD, are that reliable identification of target compounds could be achieved by accurate mass measurements along with characteristic retention time, and the great enhancement in selectivity and sensitivity allows identification and quantification of low levels of constituents in complex Polyporus umbellatus matrixes. Copyright © 2009 John Wiley & Sons, Ltd.     

Investigation of natural dyes occurring in historical Coptic textiles by high-performance liquid chromatography with UV-Vis and mass spectrometric detection

Liquid chromatography (LC) combined with ultraviolet-visible (UV-Vis) and mass spectrometric (MS) detection was utilized to study the chemical components present in extracts of natural dyes originating from fiber samples obtained from Coptic textiles from Early Christian Art Collection of National Museum in Warsaw. Chromatographic retention, ionization, UV-Vis and mass spectra of twenty selected dye compounds of flavanoid-, anthraquinone- and indigo-types were studied. Most of the investigated compounds could be ionized by positive and negative ion electrospray ionization. Difficulties with the ionization by electrospray were experienced for indigotin and brominated indigotins, but these were ionized by atmospheric pressure chemical ionization. Mass spectrometric detection, utilizing different scanning modes of a triple quadrupole mass spectrometer, combined with the UV-Vis detection was demonstrated to be a powerful approach to detection and identification of dyes in the extracts of archeological textiles. Using this approach the following compounds were identified in the extracts of Coptic textiles: luteolin, apigenin, rhamnetin, kaempferol, alizarin, purpurin, xanthopurpurin, monochloroalizarin, indirubin, and so the type of dye that was utilized to dye the textiles could be identified. Detection capabilities for several dye-type analytes were compared for the UV-Vis and mass spectrometric detection. The signal-to-noise ratios obtained for luteolin, apigenin, and rhamnetin were higher for the MS detection for most of the examined sample extracts. Purpurin, alizarin, and indirubin showed similar signal-to-noise ratios for UV-Vis and mass spectrometric detection.     

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.     

Identification of anthraquinone markers for distinguishing Rubia species in madder-dyed textiles by HPLC

The anthraquinone components of the roots of various species of madder (Rubia spp.) have been used for millennia as red colorants in textiles, carpets and other objects. Although many species of Rubia are known, only a few of them have been used widely for dyeing. Furthermore, though nearly 70 anthraquinones have been found in Rubia species, only a few of these occur consistently at relatively high levels. Knowledge of the plant dyestuffs is helpful for establishing the location of production, production method and/or history of the dyed object. Using plant material and dyed textile fibers obtained from a number of sources, and HPLC with photodiode array and mass spectrometric detection for analysis, we have been able to identify marker anthraquinones that permit differentiation of the more common species of madder used for dyeing in Eurasia. Textiles dyed with all of the species examined contain varying amounts of purpurin, but only those dyed with Rubia akane contain large amounts of 6-hydroxyrubiadin (1,3,6-trihydroxy-2-methylanthraquinone) or its glycosides. Textiles dyed with R. tinctorum contain primarily alizarin, whereas those dyed with R. cordifolia and R. peregrina contain mostly purpurin, munjistin and pseudopurpurin, but little or no alizarin or 6-hydroxyrubiadin. The latter two species cannot reliably be distinguished from each other, however. The plants themselves often contain glycoside precursors not usually seen in the dyed materials.

HPLC–DAD–MS analysis of dyes identified in textiles from Mount Athos

Organic colorants contained in 30 textiles (16th to early 20th century) from the monastery of Simonos Petra (Mount Athos) have been investigated using high-performance liquid chromatography equipped with diode-array detection and mass spectrometry (HPLC–DAD–MS). The components of natural dyes identified in samples treated by the standard HCl dyestuff extraction method were: alizarin, apigenin, butein, carminic acid, chrysoeriol, dcII, dcIV, dcVII, ellagic acid, emodin, fisetin, flavokermesic acid, fustin, genistein, haematein derivative (Hae′), indigotin, indirubin, isoliquiritigenin, isorhamnetin, kaempferide, kaempferol, kermesic acid, luteolin, naringenin, purpurin, quercetin, rhamnazin, rhamnetin, sulfuretin, and type B and type C compounds (last two are markers for Caesalpinia trees). Early, semi-synthetic dyes, for example indigo carmine, fuchsin components, and rhodamine B were identified in objects dated late 19th to early 20th century. A dyestuff extraction method which involves use of TFA, instead of HCl, was applied to selected historical samples, showing that the mild method enables efficient extraction of weld (Reseda luteola L.) and dyer’s broom (Genista tinctoria L.) glycosides. The marker compound (Hae′) for logwood (Haematoxylum campechianum L.) identification after treatment with HCl was investigated by liquid chromatography coupled to mass spectrometry (LC–MS) in negative electrospray ionization (LC–MS-ESI⁻) mode. LC–MS in negative atmospheric pressure chemical ionization (LC–MS-APCI⁻) mode was used, probably for the first time, to investigate cochineal (Dactylopius coccus Costa) samples. Positive electrospray ionization (LC–MS-ESI⁺) mode was used for identification of fuchsin components. Detailed HPLC–DAD studies were performed on young fustic (Cotinus coggygria Scop.) and Persian berries (Rhamnus trees).