Chapter 6

Identification of Organic Dyes byDirect Analysis in Real Time-Time of

Flight Mass Spectrometry

Jordyn Geiger,1 Ruth Ann Armitage,*,1 and Cathy Selvius DeRoo2

1Chemistry Department, Eastern Michigan University, 501 Mark Jefferson,Ypsilanti, MI 48197

2Conservation Department, Detroit Institute of Arts, 5200 Woodward Ave.,Detroit, MI 48202

*E-mail: rarmitage@emich.edu

We report here further developments in identifying organic dyecompounds in botanical materials and natural fiber textiles usingdirect analysis in real time-time of flight mass spectrometry(DART-TOF-MS). This method requires little to no samplepreparation, and analyses are completed in less than one minute.Analyses were performed on dyed cotton fibers from Traitédes Matiéres Colorantes du Blanchiment et de la Teinture duCoton, a late 19th century treatise on dye chemistry, mordants,and dying techniques. Sandalwood and turmeric dyes werereadily identified in the 128-year old cotton fibers by DART-MSwith no sample preparation. Simple in situ hydrolysis andderivatization have the potential to expand the applicability ofDART-MS to other dye compounds, including those in cutchand quercitron.

Introduction

A shared attribute across cultures and throughout history is the use of plantextracts as colorants for the dying of textiles and the painting of objects. Forexample, a 7th century B.C.E. cuneiform tablet providing instructions for dyingwool with madder and indigo to mimic the rare and expensive shellfish purple dyesspeaks to the importance of plant-derived dyes in ancient cultures (1). Becauseof their inherent fragility and susceptibility to degradation, textiles are rare in

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the archaeological record, and thus, as precious cultural heritage materials theyrequire judicious sampling and analysis. Compounding the challenges presentedby minute samples is the high tinting strength of most organic dyes, present onlyin nanogram to microgram quantities.

Very small samples containing miniscule quantities of dye require notonly sensitive instrumental methods but also preparation methods which do notcompromise the chemical integrity of the dyestuff in question.The oldest objectto date in which the presence of madder has been detected, a 4000 year oldpainted Egyptian leather quiver, was analyzed using surface enhanced Ramanspectroscopy (SERS) (2). While SERS provides both high sensitivity and requiresonly miniscule samples, the technique requires the preparation of colloids,hydrolysis pretreatment of the dye-mordant complex, and appropriate plasmonconditions for the Raman scattering effect to occur. High performance liquidchromatography (HPLC) is the more common method for identifying dyestuffs inhistoric textiles, though it, too, requires significant sample preparation in additionto often larger samples than are needed for SERS. Both diode array (DAD) (3–5),and mass spectrometry (MS) (6–10) detection methods have been used to identifydyes in historic and archaeological textiles.

An extensive review in 2010 described the many applications of spectroscopicmethods to the characterization of organic dyes in cultural heritage materials(11). Several methods of direct mass spectrometry are described, including highresolution laser desorption-MS applications (both with and without a matrix) (12,13). More recently, we reported on high resolution time of flight MS with directanalysis in real time (DART) ionization of indigoid, flavonoid, and anthraquinonedye materials (14). This method required no sample preparation, and possessedthe requisite sensitivity to identify organic colorants in less than 1 minute. Wereport here on the extension of that work to the detection of curcuminoids fromturmeric, pterocarpans from sandalwood, flavonols from black oak, and catechinsand tannins from cutch.

Cardon (1) provides an excellent review of the history and chemistry ofnatural dyes, which we summarize here. Turmeric, derived from the roots ofCurcuma longa, has high tinting strength, but is not particularly light fast. Thecurcuminoids (Table I) collectively are known as Natural Yellow 3. Dyes derivedfrom turmeric have long been an important part of Hindu culture, and the plantlikely originates from India. Sandalwood, too, originates from India, and hasbeen traded since medieval times, though its use in dyeing dates only to the 17thcentury. The colorants from sandalwood are considered insoluble, requiringan organic solvent for their use in dyeing. The santalins (A, B, and C) are themajor colorants, though isoflavones, pterocarpans, flavones, and aurones arealso present. Quercitron, derived from the inner bark of the black oak (Quercusvelutina), was introduced commercially in the late 18th century. The lemon yellowcolor obtained from quercitron derives primarily from quercitrin, the rhamnosideof the aglycone quercetin. Obtained from Acacia catechu, cutch and catechuare different preparations of the heartwood. The brown color of cutch comesprimarily from tannins derived from the flavonol catechin.

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Table I. Description of samples investigated

Dye material Colorant compound(s) Dye Forms Analyzed

Turmeric CurcuminDemethoxycurcuminBisdemethoxycurcumin

Powder, newly-dyed cotton, Frenchcotton (jaune de curcuma)

Sandalwood Santalin ASantalin BPterocarpinHomopterocarpin

Powder, newly-dyed cotton, Frenchcotton (rouge au santal and brun ausantal)

Quercitron Quercetin Powder, powder + formic acid,newly-dyed cotton, French cotton(jaune de quercitron)

Cutch CatechinEpicatechin

Powder, powder + formic acid,newly-dyed cotton, French cotton(light cachou, dark cachou, et boisjaune, de Laval)

Materials and Methods

Cotton standards from TestFabrics (either as skeins or woven textile) weredyed with and without alum mordant (15). Sandalwood was extracted in aminimal volume of absolute ethanol, and further prepared in deionized wateras described by Cannon (15). All other dyes were prepared in deionized water.Turmeric was obtained from a local spice shop (By the Pound, Ann Arbor,Michigan). Sandalwood and cutch were obtained from Kremer Pigments (NewYork). Quercitron bark was obtained from Maiwa Supply (Vancouver, BC).Single fibers dyed with known colorants were obtained from Traité des MatiéresColorantes du Blanchiment et de la Teinture du Coton, a late 19th century treatiseon fiber, dye chemistry, mordants, and dying techniques that includes an appendixof dyed cotton skeins. These fibers were less than 0.5 cm in length and weighedapproximately 1 mg.

Analyses were carried out on a JEOL AccuTOF mass spectrometer (JEOLUSA, Peabody, MA) equipped with a DART ionization source (Ionsense, Saugus,MA). Samples were run in positive ion mode with helium as the DART gas at aflow rate of 2.5 L/min at 300 °C. Grid voltage was set at +350 V, with orifice 1at 30 V and 120 °C. Orifice 2 and the ring lens voltage were both held at 5 V,and the peaks voltage was held at 1500 V. Each sample was calibrated by runningPEG-600 in methanol during the acquisition.

Fibers were analyzed by placing them directly into the gap between theDART source and the mass spectrometer orifice using forceps. Dye solutions andpowders, including finely ground botanical barks, were introduced on the closedend of a melting point capillary tube. To determine if acid hydrolysis affected theobserved signal, quercitron bark and cutch were treated with a few microliters of80% formic acid immediately prior to exposure to the DART source, directly on

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the melting point capillary. Mass resolution of the AccuTOF was approximately6000. Ions were observed at M+H+ in positive mode; the DART ionizationprocess has been described elsewhere (16, 17).

Results and Discussion

The primary colorant in turmeric, curcumin was observed in turmeric powderand freshly-dyed cotton as the protonated ion, at m/z 369.134 Da (Table II).Both demethoxy- and bisdemethoxycurcumin were also observed at the expectedmasses, (339.123 and 309.113 Da, respectively), at lower abundance. The128-year-old French sample was a paler yellow color than the newly-dyed cotton.However, a peak at the exact mass of curcumin was observed at approximately30% abundance. The other curcuminoids were also identified, at 12 and 16%abundances (Figure 1). No sample preparation was necessary.

Table II. Exact masses and expected DART-MS

Colorant compound(s) Formula Exact mass(M+), Da

Exact mass(M+H+), Da

Curcumin C21H20O6 368.126 369.134

Demethoxycurcumin C20H18O5 338.115 339.123

Bisdemethoxycurcumin C19H16O4 308.105 309.113

Santalin A C33H26O10 582.153 583.160

Santalin B C34H28O10 596.168 597.176

Pterocarpin C17H14O5 298.084 299.092

Homopterocarpin C17H16O4 284.105 285.113

Quercetin C15H10O7 302.043 303.050

Quercitrin C20H21O11 448.101 449.101

Catechin C15H14O6 290.079 291.087

Catechin gallate C22H18O10 442.090 443.098

Analysis of ground sandalwood bark showed no evidence of thesantalins under these analytical conditions. However, both pterocarpin andhomopterocarpin, also as M+H+ ions, were readily observed in both the barkand the dye solution. The French treatise included two samples dyed withsandalwood: rouge au santal (sandalwood red) and brun au santal (sandalwoodbrown). The two pterocarpin compounds were observed in both French samples,in ratios similar to that of the raw sandalwood bark (Figure 2).

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Quercetin was previously identified using DART-MS in textiles dyed withyellow onion skin (14). While the glycoside quercitrin reportedly predominates inthe black oak bark, only the aglycone quercetin was observed in mass spectrum.However, cotton dyed with the bark extract yielded only a small signal (~9%abundance) for quercetin, similar to that observed in the jaune de quercitronsample from the French treatise (Figure 3). The low signal intensity is clearevidence of the tinting strength of this dye.

Others have utilized formic acid to extract dye colorants from fibers throughacid hydrolysis for LC-MS studies (18). A fragment of quercitron bark was treatedwith a few drops of 80% formic acid, and introduced into the DART source,yielding a stronger signal for quercetin. This is most likely due to the hydrolysisof quercitrin to form the aglycone quercetin. Treating the quercitron-dyed cottonfibers with formic acid had no effect on the intensity of the quercetin signal.

The base peak from DART-MS analysis of the cutch powder was observedat m/z 291.084, which differs by 0.003 Da from the exact mass for protonatedcatechin. Cotton freshly-dyed with the Kremer cutch powder showed a smallpeak for protonated catechin only when the temperature of the DART gas wasincreased to 400 °C, resulting in significant charring of the fiber. The Frenchtreatise contained four different cutch-dyed cotton samples: a light khaki (cachou),dark brown (also labeled cachou), cachou et bois jaune, and cachou de Laval.Only the lightest color sample contained any indication of catechin in the massspectrum, at low abundance and higher mass difference (~12 mDa) than observedfor the standards. The primary colorant of old fustic (bois jaune, or yellowwood) isthe flavonoid morin. The French sample dyed with cutch and old fustic containedneither catechin nor morin. Using N-methyl-N-trimethylsilyl-trifluoroacetamide(MSTFA), pure catechin gallate can be directly silylated in the DART source at300°C, yielding a strong signal at 947.345 Da (R. Cody, personal communication,2011). This in situ derivatization was reportedly simple, fast, and as such hassignificant potential for colorant compounds, including tannins, that have so farbeen difficult to ionize with DART.

Figure 1. DART mass spectrum showing curcuminoids present in French textile.

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Figure 2. DART mass spectrum of sandalwood-dyed French cotton samples.

Figure 3. DART mass spectra of quercetin in cotton-dyed with quercitron bark.

Conclusions

DART-MS is a rapid and accurate method for identifying some organiccolorants in textiles without any sample preparation. Of the samples investigated,turmeric and sandalwood were successfully identified without additional samplepreparation. In situ acid hydrolysis increased the quercetin signal from quercitronbark, but had no effect on the dyed textile, possibly indicating that the textile didnot contain significant quantities of the glycoside, quercitrin. In situ derivatization,particularly silylation, has potential for identifying tannins by DART-MS.

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Acknowledgments

The authors gratefully acknowledge financial support from the NationalScience Foundation (Award MRI-R2 #0959621), as well as the EMU ChemistryDepartment and Provost’s Office. Thanks also to Robert Cody, JEOL U.S.A., forhis help with the silylation of catechin gallate.

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