This document details the synthesis and characterization of natural anthraquinone dyes using microwave-assisted methods and direct analysis in real time (DART) mass spectrometry. The study highlights the advantages of DART-MS over traditional methods, offering rapid and minimally-destructive analysis, thus enabling the identification of dye mixtures in archaeological textiles. Additionally, the synthesis process and unique fragmentation patterns of various dyes are discussed, providing insights into historical dye sources and their applications in archaeological studies.

1. Natural anthraquinone dyes and dye mixtures:
Microwave synthesis and characterization by direct analysis in real time (DART) mass spectrometry
Steven Augustin, Timothy Friebe, and Ruth Ann Armitage
Department of Chemistry, Eastern Michigan University, Ypsilanti, MI, USA 48197
Introduction
Methods
Natural anthraquinones are compounds found in plants and insects that were used as dyestuffs in
antiquity [1,2]. Although the identification of these compounds is well documented utilizing LCMS,
the extraction method destroys the sample and the conditions are harsh enough to chemically alter
the anthraquinones present [1-4]. Use of Direct Analysis in Real Time (DART) mass spectrometry
for fast, accurate and minimally-destructive testing has been embraced by forensic sciences for
many analyses, which can be applied to items of archaeological interest [5-8].
• Utilizing DART-MS for archaeological samples
• HPLC and LCMS require acid hydrolysis of textile fibers with substantial sample preparation and long run times
[1-4].
• In nearly all cases standard fibers must be prepared [1].
• DART-MS has been utilized to identify non-regioisomeric dye components on textile fibers with an analysis under
one minute requiring no sample preparation [8].
• Create a dye spectral database
➢ A validated spectral database has been created for pharmaceuticals, encompassing the entire SWGDrug list
(387 compounds) [7]
➢ Herein we apply this technique to natural dyes, utilizing statistical analysis if necessary.
• Natural anthraquinones were selected along with other dyes for MS analysis
• Identification of unique fragments are key to identifying the dye within a mixture, because many dye compounds
are identical mass regioisomers.
• Other compounds of interest included flavones and indigoid dyes.
• Some compounds are not commercially available and require synthesis.
• Synthesis based on two previously developed methods [9,10]
• Microwave-assisted Friedel-Crafts reaction using solvent
• Solventless salt melt Friedel-Crafts reaction using oil bath heating
• Utilizing salt melt in microwave will hasten reaction and eliminate use of C2H2Cl4 as a solvent
• Dyes compounds synthesized: xanthopurpurin, rubiadin, munjistin
• Individual Tests of Dye Standards
• DART-ToF-MS
• Each dye compound was tested by applying the solid or solution to the closed end of a
melting point tube
• The mass spectra were collected in negative ion mode, utilizing a protocol that rapidly
switches Orifice 1 of the ToF-MS between -30 and -90V. Positive ion mode analysis was
performed as needed.
• Dye structures in this study:
Conclusions
The synthesis requires further optimization, most importantly greater temperature control to increase yields.
The microwave-assisted solventless system reduces reaction times by approximately 75% and removes the
requirement for toxic solvents.
Utilization of DART-ToF-MS for identification of a mixture of dyes within a sample is possible due to the
unique fragments identified from each standard individually. When applied to Relbunium root samples, a
species used for dying textiles, a distribution of fragments were present that could be correlated to the dyes
in the study. Furthermore, if one knows the distribution of natural dyes within a plant source, it may be
possible to identify the plant used to dye objects of historical significance.
References
Results: Synthesis
Results: Analysis of Dye Compounds with DART-ToF-MS
1. Wouters, J.; Rosario-Chirinos, N.; J. Am. Inst. Conserv. 1992, 31, 237 – 255.
2. Szostek, B.; Orska-Gawrys, J.; Surowiec, I.; Trojanowicz, M., J. Chromatogr. A.
2003, 1012, 179 – 192.
3. Zhang, X.; Laursen, R. A., Anal. Chem. 2005, 77, 2022 – 2025.
4. Ferreira, E. S. B.; Quye, A.; McNab, H.; Hulme, A., N. Dyes Hist. Archaeol. 2002,
18, 63 – 70.
5. Steiner, R. R.; Larson, R. L., J. Forensic Sci., 2009, 54, 617-622.
6. Easter, J. L.; Steiner, R. R., Forensic Sci. Int. 2014, 240, 9 – 20.
7. Sisco, E.; Dake, J.; Bridge, C., Forensic Sci. Int. 2013, 1, 160 – 168.
8. Selvius De Roo, C.; Armitage, R. A., Anal. Chem. 2011, 83, 6924 – 6928.
9. Takano, T.; Kondo, T.; Nakatsubo, F., J. Wood. Sci. 2006, 52, 90 – 92.
10.Saha, K.; Lam, K. W.; Abas, F.; Hamzah, S.; Stanslas, J.; Hui, L. S.; Lajis, N. H.,
Med. Chem. Res. 2013, 22, 2093 – 2104.
The solventless microwave synthesis was first developed for xanthopurpurin as this
was not commercially available and necessary for our group’s studies on Galium
and Relbunium red plant dyes.
The following program was used:
• Ramp 1 minute to 120 ºC, hold 5 minutes
• Ramp 1 minute to 180 ºC, hold 5 minutes
• Cool, add acid and heat 1 minute at 100 ºC
A large temperature spike occurs when the solid phase begins to melt, causing charring. Phthalic
anhydride is prone to sublimation during the spike. Product was extracted from the acid workup
with ethyl acetate.
Crude yields of product ranged from 25 – 40%. Loss is attributed to the formation of fluorescein
and fluorescein-like byproducts and the sublimation of the phthalic anhydride.
Each dye compounds analyzed exhibited a unique fragmentation pattern at 90V, shown below.
Although some of the compounds studied have identical masses and can not be
distinguished at mild conditions, utilizing DART-MS with the fast switch protocol at -30 and
-90V gives two spectra for one analysis. This identifies the molecular weight and the CID
spectra at the same time.
Unique Fragment Ions
When distinguishing compounds with a fragment overlap, a second fragment is identified that was
completely absent in one dye but prominent in the other. For xanthopurpurin, the 210.017 is
absent, while 195.020 is absent in alizarin. For genistein, 133.028 is the base peak but 117.031 is
prominent. For emodin, 225.051 is the base peak and the 241.043 has over 50% relative
abundance.
*Munjistin was found to be unstable in the MS, especially in positive ion mode. Even at mild
conditions there is a noticeable loss of water from the molecule. For archaeological samples, use
of the dehydrated mass might be of use if munjistin is prone to breakdown in the depositional
environment.
Name Xanthopurpurin Alizarin Rubiadin Purpurin
[M-H]- ion 239.017 239.017 253.051 225.040
Primary
Fragments
195.020, 211.021 210.017, 211.021 225.057 227.041
Munjistin Emodin Apigenin Genistein Indigotin
283.021, 266.029* 269.038 269.039 269.038 261.060
239.048, 266.029* 225.051, 241.043 117.031 117.031, 133.028 217.072, 233.066
Identifier fragments applied to Relbunium hypocarpium: root and dye on wool
These regioisomers are found in red dyes
obtained from a variety of plant roots.
Rubiadin species are reported to contain only
xanthopurpurin; distinguishing between these
compounds can help to identify the plant used
to dye historic fibers and textiles.
These three compounds have the same molecular formula, C15H10O5, but are indicative of very different plant species
historically used in dyeing. Both apigenin and genistein are present in dyes made from dyer’s broom (Genista tinctoria),
while only apigenin is present in yellows prepared from weld (Reseda luteola). Emodin is found in dyes made from green
buckthorn berries, also known as Avignon berries (Rhamnus saxitilis).
These two compounds are
observed in indigo dyes (Isatis
tinctoria). They are nearly always
identified through separation by
HPLC. The ratio of these two
compounds relative to each other
is indicative of the source and
preparation of the dye.
Alizarin Xanthopurpurin
Note the peak at m/z 195 in
xanthopurpurin. This loss
of CO2 (44 mass units) is
favored by the 1,3-
substitution pattern.
Emodin
Genistein
Apigenin
Indigotin
Indirubin
While the yellow dye compounds do have quite different
fragmentation patterns, those for indigotin and indirubin
are mostly the same. Differentiating the indigoids in a
mixture would be difficult.
Positive ion modeNegative ion mode
Masses are
identified by
compound:
X – xanthopurpurin
A – alizarin
R – rubiadin
P – purpurin.
Acknowledgements
• NSF MRI-R2 award #0959621
• Relbunium root samples:
Dr. Rainer W. Bussmann,
Director William L.
Brown Center, William
L. Brown Curator of
Economic Botany,
Missouri Botanical Garden
• Saltzman reference collection
Relbunium-dyed fibers: Betsy Burr,
UCLA Conservation of
Archaeological and Ethnographic
Materials