This document describes a method developed by the CHARISMA project to isolate rare colorant compounds from the plants weld (Reseda luteola L.) and madder (Rubia tinctorum L.) using preparative liquid chromatography. Several rare standards were obtained and most were identified using mass spectrometry and NMR spectroscopy. The isolated pure standards will be shared with partners in the CHARISMA project to aid in the identification of colorants in cultural heritage objects.

1. CHARISMA Cultural Heritage Advanced Research Infrastructures:
Synergy for a Multidisciplinary Approach to Conservation/Restoration
Rare compounds studies in Reseda luteola L.
and Rubia tinctorum L.
Selection of Rare Components
Art Néss Proaño Gaibor1, Susanna Bracci2, Maarten van Bommel1*,
Jo Kirby Atkinson3, David Peggie3
The most encountered colorant compounds in dyed textiles and lake pigments in cultural heritage objects
from Europe derived from weld (Reseda luteola L.) and madder (Rubia tinctorum L.) plants. Some of the
components present in these dye plants have been elucidated and are broadly available as chemical
standards; however many components in these plants are not, despite the fact, that they are often found in
these objects and play a role in the colouring mechanism.
Within a joint research of the Cultural Heritage Advanced Research Infrastructures: Synergy for a
Multidisciplinary Approach to Conservation/Restoration (CHARISMA) project, a method was developed to
isolate these rare compounds from the plants in order to study their behavior and elucidate their structure,
if possible. This method can be used to isolated pure standard components and share them with
laboratories working in the field of colorant identification. A preparative liquid chromatography system
along with a fraction collector (fig.2) was set up to isolate flavonoid and anthraquinonic components from Fig.1 Chromatogram weld (above) and madder (beneath)
concentrated weld and madder extractions. In figure 1 the selected fractions are shown. showing in coloured bars the selected fractions
Fraction Collection of Rare Standards
These samples (called fractions, fig.3) are examined for purity with a high performance
chromatography system with photodiode array detector. The fractions are hydrolyzed to identify
its aglycone (fig.4). The pure fractions are then analyzed with an electro spray ionization (ESI)
Quadrapole time-of-flight (Q-tof) mass spectrometer (fig.5) and also with nuclear magnetic
resonance (1H and 13C-NMR).
0.30 Fig.3 Collected fractions
12.626
0.22
Apigetrin Aglycone
16.006
0.20
Apigenin
0.25
0.18
Yield 98%
Absorption at 350 nm
Absorption at 350 nm
0.16
0.20
0.14
0.12
0.15
0.10
0.08
0.10
0.06
12.910
16.008
0.04
17.420
12.773
18.615
0.05
0.02
0.00
0.00
6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00
10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00
Retention time (min)
Retention time (min)
Fig.2 fraction collector Fig.4 Chromatograms of one weld fraction (L) and same fraction after hydrolysis (R) Fig.5 ESI Q-Tof with CapLC
Elucidations and future distribution
Several rare standard of Reseda luteola L. and Rubia tinctorum L. were obtained with high purity.
Most of these standards were identified with mass spectrometry fragmentation using collisional-
induced dissociation (MS/MS) in both positive and negative mode. An example is reported in the
spectrum on the right (fig.6). The obtained standards are shown below with its respective molecular
structure, those with a question mark still need to be confirmed by NMR. After complete
identification we will proceed with the distribution of these standards to the interested partners
within the CHARISMA project. This method can be also used to analyse and isolate unknown
components in other similar flavonoid and anthraquinonic dye plants. Fig.6 Weld Fraction; MS/MS negative mode Spectrum
Apigenin-6,8-di-C-glucoside Luteolin 7-O-glucoside Apigetrin Luteolin-4’-O-glucoside? Luteolin Apigenin
Luteolin-3',7-diglucoside
C27H30O15 C21H20O11 C21H20O10 R1:OH R2: O-glycose C15H10O6 C15H10O5
C27H30O16
Mw:594.1585 g/mol Mw:448.1006 g/mol Mw:448.1006 Luteolin-3’-O-glucoside? Mw:286.048 g/mol Mw: 270.053 g/mol
Mw:610.1534 g/mol
OH OH
OH
OH
g/mol R1: O-glucose R2: OH OH
R1
HO HO OH
OH
C21H20O11 OH OH
OH
HO
Mw:448.1006 g/mol R2
O OH HO
HO HO O O O O O O O O HO O HO O
O HO HO
HO O OH
HO O
O HO O
HO OH HO OH
HO O
HO OH OH O OH OH O OH O
OH O OH O
HO
OH
W1 W2 W3 W4 W5/6 W7
HO
OH O OH O W8
Xantho-purpurin? Rubiadin 3-O-primeveroside Alizarin
Ruberythric acid Lucidin 3-O-b-primeveroside Purpurin Rubiadin Nordamnacanthal?
C14H8O4 C26H28O13 C14H8O4 C15H8O5
C25H26O13 C26H28O14 C14H8O5 C15H10O4
Mw:240.2151 g/mol Mw:548.1530 g/mol Mw:240.2151 Mw:268.2255 g/mol
Mw:534.4736 g/mol Mw:564.4999 g/mol Mw:256.2145 g/mol Mw:254.2420 g/mol
O OH O OH g/mol O OH O
OH O OH
O OH O OH
O OH O O OH O OH H
O O O O OH OH OH
O OH OH
O HO
O
OH O OH OH
O
OH O This fractions result was not conclusive ,
HO OH
O OH nordanmacanthal is one atomic mass
O OH HO O HO O unit higher than the mass spectrum in
O
Fraction M2 was not elucidated. OH O O OH O positive mode. Negative mode did not
HO show any result.
M6
HO
M1a M1b OH
M2 hydrolized M3 HO
OH M4 M5 M7
Collaborating partners:
1 - Cultural Heritage Agency of the Netherlands (OCW-RCE)
2 – Institute for the Conservation and Promotion of Cultural Heritage (ICVBC-CNR), Italy
3 - The National Gallery London (NGL), United Kingdom
*Corresponding author: m.van.bommel@cultureelerfgoed.nl Cultural Heritage Agency of the Netherlands (OCW-RCE)