Microchemical Journal 115 (2014) 78–86

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Microchemical Journal
journal homepage: www.elsevier.com/locate/microc

Comparison of extraction methods for the analysis of Indigofera tinctoria

and Carthamus tinctorius in textiles by high performance
liquid chromatography
Dimitrios Mantzouris a,b, Ioannis Karapanagiotis b,c,⁎, Costas Panayiotou a
a
Aristotle University of Thessaloniki, Department of Chemical Engineering, Thessaloniki 54124, Greece
b
Ormylia Foundation, Art Diagnosis Center, Ormylia 63071, Greece
c
University Ecclesiastical Academy of Thessaloniki, Department of Management and Conservation of Ecclesiastical Cultural Heritage Objects, Thessaloniki 54250, Greece

a r t i c l e i n f o a b s t r a c t

Article history: The extractions of indigo (Indigofera tinctoria L.) and safflower (Carthamus tinctorius L.) red and yellow from wool
Received 12 November 2013 fibres are investigated using high performance liquid chromatography with photo-diode-array detection (HPLC–
Received in revised form 14 February 2014 PDA) which is frequently coupled to Mass Spectrometry (HPLC–PDA–MS) for identification purposes. The
Accepted 18 February 2014
efficiencies of dimethyl sulfoxide (DMSO) and dimethyl formamide (DMF) to extract indigo and safflower
Available online 25 February 2014
from wool, are compared for a wide range of temperature (from 40 to 120 °C) and treatment time (from 1 to
Keywords:
30 min). Extraction procedures are investigated by measuring integrated HPLC peak areas of selected dyestuff
HPLC components, evaluating simultaneously the effects of solvent (DMSO and DMF), treatment temperature (T)
Indigo and time (t). Thus, cross-influence of the different extraction parameters (solvent, T and t) is taken into account.
Safflower Indigotin, isatin and indirubin are monitored to evaluate the extraction of indigo. Carthamin, a decomposition
Dye product of carthamin, apigenin, safflomin A, 6-hydroxykaempferol-3-O-β-D-glucoside, anhydrosafflor yellow
Cultural heritage Ct1 and Ct4 are monitored to investigate the extractions of safflower red and yellow. Overall, it is reported that
DMSO gives better extraction yields than DMF.
The best conditions for the extraction of the marker compounds of the three dyes are summarized as follows. For
indigotin, treatments of the wool with DMSO at 80 °C for t N 5 min or at 120 °C min for t = 1 min give the best
yield. Longer treatment (t N 1 min) at 120 °C results to decomposition of indigotin. For carthamin, treatments of
the wool with DMSO at 80 °C for t N 5 min or at 120 °C min for short t (b5 min) give the best yield, considering
that longer (N 5 min) treatment at 120 °C results to decomposition of the marker compound. Finally, for safflomin
A treatments of the wool with DMSO at 100 °C for t N 10 min or at 120 °C min for t = 5 min give the best result.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction
The analysis and identification of natural organic colourants
contained in historical or archaeological textiles are usually achieved
using high performance liquid chromatography (HPLC) coupled to
photo-diode-array (PDA) detection [1,2]. Mass Spectrometry (MS) con-
nected usually after the PDA has been also employed enhancing thus
the analytical capabilities of the hyphenated technique [3,4]. Methods
that do not involve a separation step such as for instance surface-
enhanced Raman spectroscopy [5] and non-invasive techniques [6]
have been occasionally applied. However, HPLC is still the most widely
adopted method for the identification of dyes because it has two
⁎ Corresponding author at: Department of Management and Conservation of
Ecclesiastical Cultural Heritage Objects, University Ecclesiastical Academy of Thessaloniki,
N. Plastira 65, Thessaloniki 54250, Greece. Tel.: +30 2310 397730; fax: +30 2310 300360.
E-mail address: y.karapanagiotis@aeath.gr (I. Karapanagiotis).
major advantages. (i) First, separation obtained with HPLC prior to
identification is extremely useful, as dyes contain several compounds
with very similar chemical structures. The direct identification of these
dyestuff components, without separation, is extremely difficult. Fur-
thermore, mixtures of several colourants have been often used in the
past to dye, for instance, textiles. (ii) HPLC provides (semi-) quantitative
data for the compounds detected in a historical/archaeological sample.
This can be extremely important to identify the exact biological source
of a dye, as was shown, for instance, for madder [7], cochineal [8,9],
dragon's blood [10] and Tyrian purple [11,12] species.
Prior to HPLC analysis, textile samples have to be treated to extract
and solubilise the contained dyes. Extraction of mordant dyes
(e.g. anthraquinones and flavonoids) can be achieved through acid
treatment, which disrupts the bond between the organic dyestuff
components and the metal cation. In 1985 a harsh dyestuff extraction
method was devised which involved the use of the strong hydrochloric
acid (HCl) [1]. In the last decade, milder methods have been developed
to extract mordant dyes, using various acids including for instance

http://dx.doi.org/10.1016/j.microc.2014.02.010
0026-265X/© 2014 Elsevier B.V. All rights reserved.
 D. Mantzouris et al. / Microchemical Journal 115 (2014) 78–86
formic [13–16], oxalic [15–18], hydrofluoric [16,19], citric [15,17] and
trifluoroacetic [15] acids. Consequently, the extraction of mordant
dyes from textile- and other-substrates has received considerable atten-
tion [13–19].
On the contrary, the extraction of vat and direct dyes, which are
attached to the textile fibres without the use of a mordant metal, has
not been investigated in detail, except probably for Tyrian purple [12].
For fibres treated with vat or direct dyes, the use of an acid is unneces-
sary, or sometimes harmful for the colourants, due to degradation
effects [2,15,20–23] and therefore treatment with a simple solvent is
usually recommended to extract the colouring materials. Dimethyl
sulfoxide (DMSO) and dimethyl formamide (DMF) are good solvents
for most of the historical vat and direct dyes and were therefore
suggested and used in previously published reports [2,20–24]. In the
case of a historical or archaeological sample, where the types of dyes
(mordant, vat, and direct dyes) present are unknown beforehand, treat-
ment of the sample with DMSO or DMF, prior to acid hydrolysis, is an
alternative route to avoid degradation of colourants which are sensitive
to acidic conditions [21,23,25]. In all these previous reports [2,20–25]
the extraction conditions which were applied, such as treatment tem-
perature and time, were selected either arbitrarily or after some very
preliminary observations. Furthermore, the extraction efficiencies of
DMSO and DMF were not compared in detail.
The goal of the present study is to compare the efficiencies of DMSO
and DMF to extract indigo, Indigofera tinctoria L. (vat dye) and safflower
red and yellow, Carthamus tinctorius L. (direct dye) from wool fibres
using HPLC. The important role of indigo and safflower red in historical
textiles is well documented [2,26]. The role of safflower yellow in
textiles of the cultural heritage is not clear [26]. However, its value in
the food industry is well known [27,28]. Attention is mainly focused
on the extractions of indigotin, carthamin and safflomin A (Fig. 1),
which are the marker compounds for the identifications of indigo,
safflower red and safflower yellow, respectively. Furthermore, other
compounds detected in the dyestuff extracts are monitored and includ-
ed in the study. It must be stressed that optimisation of the extraction
procedures is carried out, by measuring the recoveries of dyestuff com-
ponents, evaluating simultaneously the effects of solvent (DMSO and
DMF), treatment temperature (T) and time (t). Thus, cross-influence
Indigotin
Safflomin A
Fig. 1. Structures of indigotin, carthamin and safflomin A, which are the marker compounds for the identifications of indigo, safflower red and safflower yellow, respectively.
79
of the different extraction parameters (solvent, T and t) is taken into
account, as these are not investigated separately. This exhaustive inves-
tigation provides a solid framework to identify the optimised conditions
for the extractions of indigo and safflower from wool substrates. Finally,
decomposition processes of the aforementioned marker compounds
which occur after extensive heating are revealed and discussed herein.
DMSO and DMF are selected for the detailed comparative investiga-
tion, because they were previously used to treat vat and direct dyes and
were furthermore recommended for the treatment of unknown histor-
ical/archaeological samples prior to acid hydrolysis [2,20–25]. In our
initial preliminary experiments, the extraction efficiencies of pyridine
and ethyl acetate were also evaluated. However, these two solvents
were eliminated from the subsequent detailed study, as the preliminary
results showed that both DMSO and DMF provide clearly better extrac-
tion yields. This finding is in agreement with previously published, brief
investigations [29].
2. Experimental
2.1. Materials
Isatin, indigotin, apigenin, kaempferol (Fluka), carthamin (Safflower
Pigment Institute of Tianjin) and indirubin (synthesised previously
[12]) were available in pure form and were used as standard materials
for identification purposes. Wool samples dyed with indigo, safflower
red and safflower yellow were prepared within the framework of the
European project CHARISMA.
HPLC-grade dimethyl sulfoxide (DMSO, Sigma-Aldrich) and N,N-
dimethylformamide (DMF, Lab-Scan) were used to optimize the dye-
stuff extraction methods. HPLC was operated using HPLC-grade water
(Chem-Lab), HPLC-grade acetonitrile (CH3CN, J.T. Baker), trifluoroacetic
acid (TFA, Riedel-de Haën) of 99% purity and pro analysis (98–100%
purity) formic acid (HCOOH, Merck).
2.2. Dyestuff extraction methods
Dyed wools were cut into small pieces. Around 2 mg of wool dyed
with indigo was added in 500 μl of solvent (DMSO or DMF). The solvent
Carthamin
80 D. Mantzouris et al. / Microchemical Journal 115 (2014) 78–86

bath was adjusted in a reflux system and heated at various temperatures Table 1
(T) as follows: 40, 80, 100 and 120 °C were used for treatment with UV–Vis and mass spectrometric (MS) data of compounds identified in DMSO and DMF
extracts of wools dyed with indigo, safflower red and yellow.
DMSO and 80, 100 and 120 °C for DMF baths. Treatment time (t) varied
from 1 to 30 min. After heating, the sample was centrifuged at 4000 rpm Component Abbreviation UV–Vis maxima (nm) MS [M–H]−
for 1 min and the supernatant was subjected immediately to HPLC. m/z

The same protocol was followed for the analyses of safflower red and Wool dyed with indigo (Indigofera tinctoria L.)
yellow. However, the initial sample and solvent amounts were slightly Isatin IS 221, 241, 299, 415 146
Indigotin IND 215, 241, 285, 330, 605 261
different, as 3 mg of wool dyed with safflower, either red or yellow,
Indirubin INR 217, 239, 289, 363, 539 261
was added in 200 μl of solvent (DMSO or DMF).
Wool dyed with safflower red (Carthamus tinctorius L.)
Decomposition product of CAR B 219, 249, 399 449
2.3. Chromatography
Decomposition product of CAR A 247, 401 477
Carthamin CAR 219, 243, 373, 517 909
The HPLC–PDA system (Thermoquest) consisted of a 4000 quaternary Mixture of Ct compounds As described below, for safflower yellow
HPLC pump, a SCM 3000 vacuum degasser, an AS3000 auto sampler Apigenin AP 221, 267, 337 269
with column oven, a Rheodyne 7725i injector with a 20 μl sample Kaempferol KF 223, 265, 365 285

loop and a photo-diode-array detector UV 6000LP. Analyses were
carried out with an Alltima C18 (Alltech Associates) column (5 μm
particle size, 250 mm × 3.0 mm) at stable temperature of 35 °C. The
following wavelengths were selected for monitoring: 254 nm for all
dyes and, furthermore, 540 nm for indigo, 520 nm for safflower red
and 400 nm for safflower yellow. For gradient elution two solvents
were used consisting of A: 0.1% (v/v) TFA in water (pH = 2) and
B: 0.1% (v/v) TFA in CH3CN. The flow rate was 0.5 ml min−1. The follow-
ing gradient programmes were developed and used. For the analysis of
indigo: 0–1 min: linear gradient from 10% B to 50% B; 1–7 min: linear
gradient to 60% B; 7–9 min: linear gradient to 90% B; 9–11 min: 90% B
isocratic. For the analysis of safflower red: 0–6 min: linear gradient
from 5% B to 30% B; 6–11 min: linear gradient to 60% B; 11–14 min:
60% B isocratic; 14–16 min: linear gradient to 70% B; 16–17 min: linear
gradient to 95% B. For the analysis of safflower yellow: 0–5 min: linear
gradient from 5% B to 25% B; 5–11 min: linear gradient to 30% B; 11–
15 min: linear gradient to 60% B; 15–17 min: 60% B isocratic; 17–
20 min: linear gradient to 95% B.
For the comparative study, HPLC–PDA analyses of dyed wools were
carried out in triplicate and mean values of the HPLC integrated areas
were calculated for the monitored compounds. These were normalized
taking into account the masses of the tested wool samples. Relative
standard deviations for the three marker compounds such as indigotin,
carthamin and safflomin A were on the order of 7, 5 and 5%, respectively.
Relative standard deviations for compounds detected in smaller amounts
were as follows: isatin 9%, indirubin 6%, decomposition product of
carthamin A 9%, apigenin 9%, 6-hydroxykaempferol-3-O-β-D-glucoside
9%, anhydrosafflor yellow B 8%, compound Ct1 8% and compound Ct4
9%.
HPLC–PDA was connected to Mass Spectrometry (HPLC–PDA–MS)
to identify components of safflower red and safflower yellow, not avail-
able in pure form. A mass spectrometer (Thermoquest) equipped with
an ESI interface which was operated in the negative ionisation mode
and a single quadrupole mass filter was used to record ion signals. The
ESI probe temperature was 400 °C and corona and cone voltages were
4.0 kV and 30 V, respectively. Chromatography was carried out using
the same column and conditions described in the previous paragraph,
except for the gradient elution programme and solvents, which had to
be changed as TFA is an aggressive chemical for the MS detector and
furthermore results in signal suppression [30,31]. For this reason, formic
acid and CH3CN were used, as described elsewhere [32].
3. Results and discussion
Table 1 summarizes the UV–Vis chromatographic and mass spectro-
metric data of the components which were identified in DMSO and DMF
extracts of the dyed wools. According to the Experimental, isatin, indi-
gotin, indirubin, carthamin, apigenin and kaempferol were available in
pure forms. Other compounds included in Table 1 were identified by
comparing our HPLC–PDA–MS results with corresponding data of the
literature [20,27,28,33–35]. In particular, the Ct1, Ct2, Ct3 and Ct4
Wool dyed with safflower yellow (Carthamus tinctorius L.)
Safflomin A SAF A 237, 345, 377, 399 611
6-Hydroxykaempferol-3-O-β- KF-GLU 221, 275, 339 463
D-glucoside
Anhydrosafflor yellow B SAF B 223, 243, 345, 409 1043
Compound Ct1 Ct1 223, 269 582
Compound Ct2 Ct2 221, 277 582
Compound Ct3 Ct3 223, 291 582
Compound Ct4 Ct4 227, 297, 307 582
Carthamin As described above, for safflower red
compounds, contained in C. tinctorius, were identified by comparing
our results with corresponding UV–Vis and mass spectra data [20].
The chemical structures of these compounds are unknown. However,
they can be used as markers for the identification of safflower red
when the standard HCl method is applied for dyestuff extraction be-
cause CAR, the actual marker component of the red dye, decomposes
in strong acidic conditions [20]. It must be stressed that the Ct com-
pounds were detected in the DMSO and DMF extracts of both yellow
and red wools dyed with safflower. However, the Ct compounds were
separated sufficiently only with the gradient programme used to ana-
lyse safflower yellow. Similarly, CAR was detected in the extracts of
both yellow and red wools dyed with safflower, but in very low and
high amounts, respectively. HPLC peak areas of CAR were integrated
and measured only for the extract of safflower red.
Decomposition products of CAR were recently revealed and
described by Laursen and Mouri [33]. Two CAR derivatives, labelled as
A and B in Table 1 according to Laursen and Mouri [33], were identified
in our study. Finally, components of safllower yellow such as safflomin
A, 6-hydroxykaempferol-3-O-β-D-glucoside and anhydrosafflor yellow
B were identified using UV–Vis and mass spectra reported elsewhere
[27,28,34,35].
3.1. Extraction of indigo
Fig. 2 displays a typical chromatogram for wool dyed with indigo.
The following compounds are identified: IS, IND which is the marker
compound for the identification of indigo, and INR. Fig. 3 shows inte-
grated HPLC peak areas of these dye components measured at 254 nm
after their extraction from dyed wool fibres using DMSO or DMF as a
function of treatment time (t) at various temperatures (T).
The results collected for IND (Fig. 3a) suggest that the best extraction
yield for the main colouring component of indigo is obtained when the
wool sample is treated with DMSO at 80 °C for more than 5 min.
Alternatively, heating the DMSO extracting bath at 120 °C but only for
1 min gives also a comparable-good yield for IND, demonstrating thus
the cross-impact effects of treatment t and T: long t is necessary to
achieve a good extraction yield of the dye when low T (=80 °C) is ap-
plied for sample treatment, whilst shorter t must be considered when
high T (=120 °C) is used.
 D. Mantzouris et al. / Microchemical Journal 115 (2014) 78–86
7000000
6000000
Absorbance (micro AU) 5000000
4000000
3000000
IS
2000000
1000000
0
5 6
Fig. 2. HPLC–PDA chromatogram collected at 254 nm for a DMSO extract of wool dyed with indigo. The following compounds are identified: isatin (IS), indigotin (IND) and indirubin (INR).
The maximum amount of IND extracted at 80 °C is constant for a
long time range of 10–30 min, where a plateau is displayed in the plot
of Fig. 3a. The best temperature for DMF extraction is 100 °C as this
gives a constant, maximum yield for a treatment period of 15–25 min.
However, DMSO/80 °C gives better extraction yield than DMF/100 °C.
Interestingly, the amount of the extracted IND decreases with t at
high T such as 120 °C. This is observed for treatment with either DMF
or DMSO; in the latter a decrease of the extracted amount of IND with
t is reported even at 100 °C. Apparently, degradation of IND is promoted
at high T and becomes a dominant process which is depicted in the
results of Fig. 3a as a decrease in the extraction yield of IND. The oxida-
tive degradation of IND, results in cleavage into smaller compounds,
including IS [36,37].
The results for IS are displayed in Fig. 3b, which shows that the
amount of this compound recorded with HPLC, increases with treat-
ment t and T. No plateau/saturation is recorded in the plot of Fig. 3b.
The increase of the amount of IS with t, is more pronounced at elevated
T where the extracted amount of IND is reduced (Fig. 3a). In particular,
the rapid increase of the amount of IS is observed at 120 °C/DMSO,
120 °C/DMF and 100 °C/DMSO (Fig. 3b). In these extraction conditions
the amount of IND decreases with t, as shown in Fig. 3a, thus suggesting
that IND decomposes to IS.
INR is more stable than IND, according to the results reported in
Fig. 3c, as no decrease in the extracted amount of this compound is
recorded with t at any T. Treatment for at least 10 min at 80 °C with
DMSO, gives the best results, which are however comparable to the
extraction yields obtained for 100 °C/DMF. Increasing T of the DMSO
bath to 120 °C, results in a decrease of the extracted amount of INR,
compared to what is extracted at 80 °C (and 100 °C). Therefore decom-
position processes of INR accelerated by heat may be involved but these
do not have the dramatic effects recorded for IND. For DMF a decrease in
the extraction yield is observed when T increases from 100 to 120 °C.
3.2. Extraction of safflower red
Fig. 4 shows a chromatogram of safflower red extract. The following
compounds are identified using the data of Table 1: compounds B and A
81
IND
INR
7 8 9 10
Time (min)
(decomposition products of CAR [33]), CAR, which is the marker
compound for the identification of safflower red, a mixture of the Ct
compounds [20], which are co-eluted with CAR when 254 nm is used
for monitoring, but are not detected at 520 nm, AP and KF. Separation
of the Ct compounds was achieved using the gradient programme
applied for safflower yellow and therefore the extraction of these com-
pounds is discussed in the next chapter (extraction of safflower yellow).
It must be noted, that monitoring of the Ct compounds is not within the
main goals of the present study. The Ct compounds are important when
harsh acidic conditions are applied to extract safflower red, as CAR
which is the actual marker compound of the dye, is destroyed by HCl
[20]. Sample treatments with DMSO or DMF, investigated in the present
study, preserve CAR. These treatments can be applied to any unknown
historical sample, prior to acid hydrolysis, avoiding thus the undesirable
degradation of CAR.
Fig. 5 shows integrated HPLC peak areas for CAR, the decomposition
product of carthamin A and AP. Product B and KF are not included in the
results of Fig. 5, because they correspond to very small HPLC peaks
(Fig. 4) and therefore their quantitation is subjected to large variations.
Also, the Ct components are excluded from the results of Fig. 5 because
they are co-eluted in mixture (Fig. 4).
The results presented in Fig. 5a show that the best extraction yield
for CAR is obtained when the wool sample is treated with DMSO at
80 °C for at least 5 min. Further treatment of the wool sample for longer
periods (at 80 °C) does not have any significant effect on the extracted
amount of CAR. Alternatively, heating the DMSO extracting bath at
120 °C gives also a good yield for CAR, provided that treatment t is
very short and within 1 to 5 min. Consequently, cross-influence of
treatment T and t is revealed by the data of Fig. 5a. Longer t (N5 min)
at 120 °C leads clearly to decomposition of CAR, as the amount of the
compound measured by HPLC decreases with time. This decrease is
not recorded at lower T such as 100 and 80 °C. Saturation behaviours
are observed for the extracted amount of CAR for treatment longer
than 5 min at 80 °C or 100 °C. However, the extraction yield of CAR
obtained at 100 °C/DMSO is lower than at 80 °C/DMSO.
Finally, Fig. 5a shows that DMSO gives overall better results
than DMF. The maximum amount of CAR extracted using DMF, is
82 D. Mantzouris et al. / Microchemical Journal 115 (2014) 78–86

3,5E+08
a) IND

HPLC peak area (micro AU)

3,0E+08
40 C (DMSO)
2,5E+08
80 C (DMSO)
2,0E+08 100 C (DMSO)

1,5E+08 120 C (DMSO)

80 C (DMF)
1,0E+08
100 C (DMF)
5,0E+07 120 C (DMF)

0,0E+00
0 10 20 30

3,0E+07
b) IS
HPLC peak area (micro AU)

2,5E+07
40 C (DMSO)
2,0E+07 80 C (DMSO)
100 C (DMSO)
1,5E+07
120 C (DMSO)

1,0E+07 80 C (DMF)
100 C (DMF)
5,0E+06
120 C (DMF)

0,0E+00
0 10 20 30

1,4E+07
c) INR
HPLC peak area (micro AU)

1,2E+07
40 C (DMSO)
1,0E+07
80 C (DMSO)
8,0E+06 100 C (DMSO)

6,0E+06 120 C (DMSO)

80 C (DMF)
4,0E+06
100 C (DMF)
2,0E+06 120 C (DMF)

0,0E+00
0 10 20 30
Treatment time (min)

Fig. 3. Integrated HPLC peak areas of compounds extracted from wool samples dyed with indigo after treatment with DMSO and DMF at various conditions (treatment time and
temperature). Measurements at 254 nm are shown for the following compounds: (a) IND, (b) IS and (c) INR.

CAR+mixture of Cts
16000000

14000000

12000000
Absorbance (micro AU)

520nm 254nm
shoulder
10000000

8000000
CAR pure CAR+Cts
6000000
11,3 Time (min) 11,8 11,3 Time (min) 11,8

4000000
AP KF
2000000 A
B

0
8,0 8,5 9,0 9,5 10,0 10,5 11,0 11,5 12,0 12,5
Time (min)

Fig. 4. HPLC–PDA chromatogram collected at 254 nm for a DMSO extract of wool dyed with safflower red. The following compounds are identified: two decomposition products of
carthamin labelled B and A, carthamin (CAR) in mixture with the Ct compounds, apigenin (AP) and kaempferol (KF). The mixture of CAR and Ct compounds is recorded when 254 nm
is used for monitoring, as shown in the right inset which is an enlargement of the CAR + Cts peak. The Ct compounds do not absorb light in the visible range (Table 1) and therefore
pure CAR is recorded at 520 nm (left inset).
 D. Mantzouris et al. / Microchemical Journal 115 (2014) 78–86 83

1,0E+08 a) CAR
9,0E+07

HPLC peak area (micro AU)

8,0E+07 40 C (DMSO)
7,0E+07 80 C (DMSO)
6,0E+07
100 C (DMSO)
5,0E+07
120 C (DMSO)
4,0E+07
80 C (DMF)
3,0E+07
100 C (DMF)
2,0E+07
120 C (DMF)
1,0E+07
0,0E+00
0 10 20 30

HPLC peak area (micro AU)

7,0E+06 b) A
6,0E+06
40 C (DMSO)
5,0E+06 80 C (DMSO)

4,0E+06 100 C (DMSO)

3,0E+06 120 C (DMSO)

2,0E+06 80 C (DMF)

100 C (DMF)
1,0E+06
120 C (DMF)
0,0E+00
0 5 10 15 20 25 30

1,0E+07 c) AP
9,0E+06
HPLC peak area (micro AU)

8,0E+06 40 C (DMSO)

7,0E+06 80 C (DMSO)
6,0E+06 100 C (DMSO)
5,0E+06 120 C (DMSO)
4,0E+06
80 C (DMF)
3,0E+06
100 C (DMF)
2,0E+06
120 C (DMF)
1,0E+06
0,0E+00
0 10 20 30
Treatment time (min)

Fig. 5. Integrated HPLC peak areas of compounds extracted from wool samples dyed with safflower red after treatment with DMSO and DMF at various conditions (treatment time and
temperature). Measurements at 520 nm are shown for (a) CAR and at 254 nm for (b) compound A and (c) AP.

independent of the tested T (80, 100 and 120 °C). The best yield obtain-
ed with DMF, however, is almost half of the corresponding result ob-
tained with DMSO (T = 80 °C for more than 5 min).
The thermal stability of CAR in aqueous solutions has been investi-
gated in the past by Kim and Paik [38] and more recently by Laursen
and Mouri [33], who suggested that carthamin hydrate undergoes a
reverse aldol condensation to give two fragments, which were labelled
A and B [33]. In Fig. 4, these compounds were identified in the extracts
of safflower red by carefully comparing the HPLC data collected herein
with the UV–Vis and mass spectrometric (MS) data reported by Laursen
and Mouri [33,39]. It must be noted, however, that with our HPLC
gradient programme, compound B is eluted before A (Fig. 4), reversing
thus the order of retention times reported in the previously published
article [33]. Another interesting point is that water, which is involved
in the suggested decomposition mechanism of CAR [33], was not inten-
tionally added to the DMSO and DMF treatment baths of our study.
However, wool absorbs water molecules from the atmosphere and
therefore small amounts of water should be present in the extraction
solutions.
Integrated HPLC peak areas for the decomposition product A are pre-
sented in Fig. 5b which shows that higher amounts of this compound
are detected in DMSO than in DMF extraction baths. The same tendency
was observed for the extraction of compound B, which however
was detected in trace (Fig. 4) and therefore it is not included in the
quantitative results of Fig. 5. The superiority of DMSO over DMF for
the carthamin decomposition products should be related to the higher
extraction yields of CAR obtained with DMSO compared to DMF
(Fig. 5a) and the subsequent degradation of CAR observed in the
DMSO baths (Fig. 5a).
Finally, Fig. 5b shows that the extracted amount of compound A
increases with temperature. This is clear for the DMSO extractions. For
the DMF baths the increase of the extracted amount of compound A
with T cannot be revealed at this scale of the y-axis.
Other compounds detected in the extract of safflower red are AP and
KF (in trace), which are contained in several plant species and are not
considered as markers for the identification of safflower. The results of
the HPLC integrated areas for AP, which was detected in considerable
amounts, are provided in Fig. 5c, which shows that DMSO and DMF
give comparable results.
3.3. Extraction of safflower yellow
Fig. 6 shows a typical chromatogram obtained for wool dyed with
safflower yellow. The following compounds are identified: SAF A, KF-
GLU, SAF B, Ct1, Ct2, Ct3, Ct4 and CAR (in trace). Fig. 7 shows integrated
HPLC peak areas of the aforementioned compounds, except for CAR,
which was measured previously for safflower red and Ct2 and Ct3
which were not separated satisfactory for peak quantitation purposes.
The results for SAF A are shown in Fig. 7a. Cross-influence effects of
treatment T and t were described previously for the extraction of IND
and CAR. Comparable, high extraction yields for the markers of indigo
and safflower red are obtained either at very high T (120 °C) and very
84 D. Mantzouris et al. / Microchemical Journal 115 (2014) 78–86
SAF A
20000000
18000000
16000000
Absorbance (micro AU) 14000000
SAF B
12000000
10000000
8000000
6000000 KF-GLU
4000000
2000000
0
8 10
Fig. 6. HPLC–PDA chromatogram collected at 254 nm for a DMSO extract of wool dyed with safflower yellow. The following compounds are identified: safflomin A (SAF A),
6-hydroxykaempferol-3-O-β-D-glucoside (KF-GLU), anhydrosafflor yellow B (SAF B), Ct1, Ct2, Ct3 and Ct4, and carthamin (CAR).
short t or at moderate T (80 °C) and longer t. A similar trend is reported
in Fig. 7a, which shows that the best extraction yield for SAF A, the
marker compound of safflower yellow, is obtained when the wool
sample is treated with DMSO either at 100 °C for at least 10 min (or
more) or at higher T (= 120 °C) for shorter t (= 5 min). The amount
of SAF A recorded by HPLC at T = 120 °C decreases with t, implying
that a decomposition process of the compound becomes dominant.
Degradation processes are monitored at 120 °C for KF-GLU and SAF B,
according to Figs. 7b and c. Several factors affect drastically the stability
of safflower yellow, which is a rather sensitive dye as described in the
literature [40–42]. Overall, Figs. 7a, b and c show that DMSO provides
considerably better extraction yields than DMF at any t or T for the
components of safflower yellow such as SAF A, KF-GLU and SAF B.
Attention is now turned to the extractions of Ct1 and Ct4 com-
pounds, described in Figs. 7d and e, respectively. The results for these
compounds are summarized as follows: (i) DMSO is a better solvent
than DMF, as it provides considerably better extraction yields for any t
or T. (ii) The two compounds appear to be stable as no decomposition
process is suggested by the data of Figs. 7d and e. (iii) When the wool
sample is treated with DMSO, T does not have any significant effect on
the extracted amounts of these Ct compounds, provided that the T is
not lower than 80 °C and t N 5 min. The curves reported in Figs. 7d
and e show that the amounts of Ct1 and Ct4 initially increase with t
and then at t N 5 min they reach maximum values which are constant.
Extraction yields obtained for t N 5 min with DMSO/80 °C, DMSO/
100 °C and DMSO/120 °C are comparable.
As described previously, systematic measurements on the extrac-
tions of Ct2 and Ct3 could not be carried out, because these two
compounds were not separated sufficiently (Fig. 6). However, rough
measurements-observations, suggested that the extractions of the Ct2
and Ct3 follow, overall, the same behaviours reported above for Ct1
and Ct4.
4. Conclusions
The efficiencies of DMSO and DMF to extract indigo (I. tinctoria L.)
and safflower (C. tinctorius L.) red and yellow from wool fibres were
Ct3
Ct4
Ct2
CAR
Ct1
12 14 16
Time (min)
evaluated and compared using HPLC–PDA–MS. The integrated HPLC
peak areas of several compounds were measured (Figs. 3, 5 and 7) to
carry out the comparative study, after treating dyed wool samples
under various conditions; treatment temperature (T) varied from 40
to 120 °C and time (t) from 1 to 30 min.
Fig. 8 summarizes the best extraction conditions recorded for
indigotin (IND), carthamin (CAR) and safflomin A (SAF A), which are
the marker compounds for the identifications of indigo, safflower
red and safflower yellow, respectively. The results described in Fig. 8
correspond to treatment with DMSO, which gave better extraction
yields than DMF and were generated from the data of Figs. 3a, 5a and
7a. Furthermore, the results of Fig. 8 were confirmed by an additional
experiment which was carried out independently, where dyed wool
samples were treated according to the conditions described in the fig-
ure. Extraction recoveries recorded with the additional experiment
(not shown) fall within the statistical error bars included in Fig. 8.
Fig. 8 reveals the cross-influencing role of t and T on the extraction of
the three compounds: longer t is necessary when relatively low T is
applied for sample treatment whilst shorter t must be considered
when higher T is used. Although treatment at low T and long t may
give slightly better yields according to the results of Fig. 8, the differ-
ences with the results reported for treatment at 120 °C (high T) and
short t are within the error bars.
It must be stressed that the use of high T (= 120 °C) may lead to
decompositions of the three marker compounds, as shown in Figs. 3a,
5a and 7a. Consequently, t must be strictly controlled when 120 °C is
selected to treat dyed wool samples. For T = 80 °C the maximum
yields for indigotin and carthamin are obtained for a very long
range of t (N 5 min). For the extraction of safflomin A, slightly higher
T (= 100 °C) is recommended at which the compound appears to be
stable for a long range of t (N 10 min).
Acknowledgements
Financial support by the 7th Framework Programme of the
European Union (CHARISMA Grant Agreement no. 228330) is gratefully
acknowledged (http://www.charismaproject.eu/). The authors would
 D. Mantzouris et al. / Microchemical Journal 115 (2014) 78–86 85

1,4E+08 a) SAF A 80oC 120oC 80oC

120oC
100oC
120oC
HPLC peak area (micro AU)

100,0

Normalized HPLC peak area

1,2E+08
40 C (DMSO)
1,0E+08
80 C (DMSO)
80,0
8,0E+07 100 C (DMSO)
120 C (DMSO)
6,0E+07
80 C (DMF) 60,0
4,0E+07

t > 10 min
t < 5 min
100 C (DMF)

t = 1 min

t = 5 min
t > 5 min

t > 5 min
2,0E+07 120 C (DMF) 40,0

0,0E+00
0 5 10 15 20 25 30
20,0
2,5E+07 b) KF-GLU
HPLC peak area (micro AU)

0,0
2,0E+07 40 C (DMSO)
Indigotin Carthamin Safflomin A
80 C (DMSO)
1,5E+07 100 C (DMSO) Fig. 8. Summary of the best extraction procedures for IND, CAR and SAF A. For each
120 C (DMSO) compound extraction yields shown in the y-axis are normalised to the highest value,
1,0E+07 taken as 100%. The conditions described (t and T) correspond to treatment with DMSO
80 C (DMF)
which gave better extraction yields than DMF. Normalized HPLC peak areas reported at
100 C (DMF)
5,0E+06 80 °C (for IND and CAR) and 100 °C (for SAF A) correspond to the plateaus of the curves
120 C (DMF) shown in Figs. 3a, 5a and 7a. The plot illustrates the cross-influence effect of t and T.

0,0E+00
0 5 10 15 20 25 30 like to thank Dr. Maarten van Bommel and Dr. Jo Kirby for providing the
dyed wool samples and Dr. Richard Laursen for useful discussions on the
7,0E+07
c) SAF B decomposition of carthamin.
HPLC peak area (micro AU)

6,0E+07
40 C (DMSO)
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