NMR spectroscopic study of the Murex trunculus dyeing process

Article abstract of DOI:10.1002/mrc.2685

It is widely accepted that indigo dyes derived from Murex trunculus were used to produce the biblical dyes tekhelet and argaman. We describe a method of following the debromination of natural leucoindigos and their binding to wool using NMR spectroscopy. Debromination is observed prior to reaction with the wool and prior to oxidation. Binding to the wool is shown to occur prior to oxidation. NMR allows the dyeing process to be followed. This, in principle, could be used to correct problems during dyeing that would increase the reliability of the process. Copyright

Full text of DOI:10.1002/mrc.2685

Research Article
Received: 22 June 2010
Revised: 25 August 2010
Accepted: 31 August 2010
Published online in Wiley Online Library: 28 September 2010
(wileyonlinelibrary.com) DOI 10.1002/mrc.2685
NMR spectroscopic study of the Murex
trunculus dyeing process
Rina C. Hoffman,a Reut C. Zilbera and Roy E. Hoffmanb∗
It is widely accepted that indigo dyes derived from Murex trunculus were used to produce the biblical dyes tekhelet and
argaman. We describe a method of following the debromination of natural leucoindigos and their binding to wool using NMR
spectroscopy. Debromination is observed prior to reaction with the wool and prior to oxidation. Binding to the wool is shown
to occur prior to oxidation. NMR allows the dyeing process to be followed. This, in principle, could be used to correct problems
c
during dyeing that would increase the reliability of the process. Copyright ꢀ 2010 John Wiley & Sons, Ltd.
Keywords: indigo; dye; tekhelet; NMR spectroscopy; leucoindigo; biblical dyes
Introduction
last few years.[13] According to Jewish law,[2] one needs to use the
correct ‘snail’ but there is no requirement to use any other specific
chemicals. Therefore the modern industrial process is religiously
valid provided that the ‘snail’ has been correctly identified.
The dried snail dye that is produced industrially contains
between 5 and 10% dye. Most of the rest is protein along
with other materials such as shell. The dye is formed when the
mollusksarebrokenopenandthecontentsoftheirhyprobranchial
glands exposed to the air.[14,15] In the modern dyeing process,
the dye is reduced with sodium dithionite (Na2S2O4) in sodium
hydroxide solution. The dye undergoes a two-electron reduction
and dissolves to yield a pale yellow solution of leuco dye (Scheme
1). If the dye is applied to the wool at this stage then a purple
color (Hebrew – argaman, Latin – purpura) is produced due to the
presence of bromoindigos. In order to obtain the biblical-blue
tekhelet the reduced solution is exposed to UV light that is known
to debrominate the reduced dye (Scheme 2).[16,17] The sensitivity
to sunlight was already known in ancient times.[18,19] Ammonium
sulfatebuffersolutionisthenaddedtoreducethebasicitysoasnot
to damage the wool. The wool is then inserted and when removed
and exposed to air the dye oxidizes to produce the tekhelet color.
The reduction and oxidation are both two-electron processes.
If any single electron reduction occurs in aqueous solution most
disproportionates to zero and two-electron reduction states. We
found that a stable partially reduced solution of indigo can
be obtained by diluting an aqueous leucoindigo solution with
acetone and exposing it to a limited amount of air. This yields
a red solution of what is presumed to be the indigo radical that
does not show any indigo or leucoindigo signals in the regular 1H
NMR spectrum. This solution does not dye wool, adding weight to
the argument that single electron reduction is insufficient for the
dyeing process.
According to Jewish tradition, the tekhelet (a blue colored) dye
was used in fringes of clothing (tsitsit) priestly garments and the
Temple furnishings. According to the Talmud, tekhelet is produced
from the ‘blood of the sea snail’.[1] The Talmud mentions a dye by
the name of kleiilan[2] (which is identified as indigo produced from
plants) that looks the same as tekhelet and was used as a cheap
alternative. However, for the religious ordinance of tsitsit this
cheaper alternative was considered invalid. According to Hertzog,
the production of tekhelet from the ‘snail’ ceased after the Arab
invasion of the Israeli coast in 638 CE. He reaches this conclusion
because the use of tekhelet is mentioned in the Talmud[1] (written
before this date) and the lack of tekhelet is noted in Midrash
Tanchuma that was written around 750 CE.[3,4]
Without tekhelet, the Jewish tradition of tsitsit has continued
usingonlywhitethreadsthroughthegenerations.Thefirstattempt
to reinstitute the use of tekhelet was made by the Radziner
Rebbe (Rabbi Gershon Leiner) in 1888. He identified a squid, Sepia
officinalis, as the ‘snail’ and produced blue threads for thousands
of his followers but his opinion was not widely accepted. His dye
was tested chemically by Hertzog and found to be Prussian blue
that was derived from chemicals added in the process of making
the dye and not from the squid itself.[5,6]
Historical studies over the past 150 years indicate that the
original source of tekhelet is the sea mollusk Murex trunculus.
Hertzog, in his PhD thesis, summarized the conclusions of his
research but concluded that the source of tekhelet was another
mollusk, Janthina.[5] A likely reason for his mistake is that the
Talmud mentions a ‘snail’ with a color similar to the sea. Hertzog
did not collect the mollusks himself but received them after they
had been cleaned of the blue/green flora that covers the living
mollusk in the sea. Therefore, he did not know what the mollusk
lookedlikeinthesea.Hisresearchremainedtheoreticalanditisnot
known if he ever attempted to produce dye from Murex trunculus.
In 1985, the first known production of tekhelet in recent times
was carried out from MurexTrunculus.[7–9] This was developed into
an industrial process for the production of tsitsit for religious use.
The truth is that the industrial process uses chemicals that were
not known in the distant past. The ancient process was based on
biochemical fermentation[10–12] that was only reproduced in the
∗
Correspondence to: Roy E. Hoffman, Institute of Chemistry, The Hebrew
University of Jerusalem, Jerusalem 91904, Israel. E-mail: Royh63@gmail.com
a
Ulpanat Zvia, 30 Derech Kedem, Ma’ale Adummim 98452, Israel
b
Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904,
Israel
c
Magn. Reson. Chem. 2010, 48, 892–895
Copyright ꢀ 2010 John Wiley & Sons, Ltd.

NMR spectroscopic study of the Murex trunculus dyeing process
O-
3
H
O
H
Reduction
Na2CO3, H2O,
Na2S2O4
4
7
4
7
R’
R’
3a
7a
3a
7a
3
2
N
N
5
5
6
2
6
N1
N1
Oxidation
O2, H2O
R
R
O-
O
H
H
R,R’ = H, Br
Scheme 1. Reduction and oxidation of indigo derivatives.
O-
H
O-
3
H
4
4
hv, H2O,
Br
3a
7a
3
3a
7a
-Br•, -•OH
N
N
5
6
5
6
2
2
N1
N1
Br
7
7
H
O-
H
O-
Scheme 2. Debromination of leucobromoindigo.
HPLC shows that the Murex trunculus dye contains, indigo, 6-
monoboroindigo, 6,6ꢁ-dibromoindigo, asmallamountofindirubin
and some bromo derivatives of indirubin.[20–23]
Argaman
(purple)
Tekhelet
(blue)
Intheancientpast, asmallamountofdyewasremovedfromthe
batch and tested by dyeing a small amount of wool with it. This is a
destructive and invasive test. Even the modern industrial method
exposes the leuco dye to daylight or sunlight for an excessive
period of time to ensure complete debromination. Sometimes,
the debromination is incomplete but the color can be turned bluer
by steaming the wool. This is possible if all the 6,6ꢁ-dibromoindigo
has been removed because heating 6-monobromoindigo causes
a change in its crystal structure and makes it turn blue.[24] NMR
spectra of luecoindigoids have only been measured in the last
decade.[25] Here we apply NMR spectroscopy to the analysis of the
reduced dye in a non-destructive manner. In this work we provide
proof that the debromination takes place during the reduced
stage and show that the debromination and absorption of the dye
can be followed spectroscopically and noninvasively.
Figure 1. The effect of exposure to light during the reduced stage on wool
dyed with Murex trunculus.
water/TMS standard. The chemical shifts of pure leucoindigo, 6-
leucobromoindigo and 6,6ꢁ-leucodibromoindigo were measured
in D2O/KOH/Na2S2O4 solution and were used for interpreting the
spectra of the batches.
Experimental
Thepresaturated1H-NMRspectraofthebatchesweremeasured
Snail dye and Na2S2O4 were provided by Ptil Tekhelet. 6-
Bromoindigo and 6,6ꢁ-dibromoindigo were provided by I. Zei-
derman. KOH, (NH4)2SO4, indigo – Sigma. D2O – CIL.
1
and one batch exposed to cloudy daylight for 1 / h. There was
2
a considerable water signal in the samples so a 5-s pre-saturation
pulse was applied to the water signal to reduce the dynamic
range. The 1H-NMR spectrum was remeasured. A D2O solution of
(NH4)2SO4 was added and the spectrum remeasured. The tubes
were inverted so that the solution made contact with the wool
for 1 h. Afterwards, the tubes were turned back and their spectra
remeasured. The wool was removed and exposed to the air for 1 h
then washed with distilled water.
The experiment is based on the industrial process (Guberman J,
private communication) with some adjustments. For the purpose
of NMR spectroscopy, D2O was used instead of regular water and
the size of the batches were scaled down by a factor of 3000
compared with industrial batches. As the surface area to volume
ratio is much larger for small batches, allowing oxygen to diffuse
into and reoxidize the dye, the batches were prepared under an
inert argon atmosphere.
Two batches were prepared in extended NMR tubes. Snail dye
(25 mg), Na2S2O4 (20 mg) and KOH (∼7 mg) were placed in each
tube and the tube flushed with argon. In a darkened room, 700 µl
of D2O was deoxygenated with argon and injected into the NMR
tube.Eachtubewasopened,wool(100 mg)insertedintotheupper
part, the tube closed and flushed again with argon. The tubes were
gently heated to below boiling until the dark solution turned pale
yellow. TMS vapor (0.1 ml) was added as a chemical shift reference.
NMR spectra were measured using a Bruker Avance II 500 MHz
spectrometer at 28.3 ◦C. The temperature was calibrated using a
Results and Discussion
The dyed wool that was the final product clearly showed that the
batch that was exposed to light was dyed tekhelet (blue) while the
batch that had not been exposed to light was argaman (purple)
(Fig. 1).
The 1H-NMR spectrum of the reduced dye shows signals arising
mainly from proteins. In the aromatic region (6.5–8 ppm) weak
signals of the dye can also be discerned by comparison with the
c
Magn. Reson. Chem. 2010, 48, 892–895
Copyright ꢀ 2010 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/mrc

R. C. Hoffman, R. Zilber and R. E. Hoffman
Table 1. Chemical shifts of leucoindigo and its bromo derivatives in
D2O/KOH/Na2S2O4
Difference (x4)
6-Bro-
moleucoindigo
6-Bro-
moleucoindigo
6, 6ꢁ-dibro-
Indigo (unsubstituted ring) (substituted ring) moleucoindigo
H-4 7.63
H-5 7.11
H-6 7.04
H-7 7.38
7.61
7.08
7.01
7.34
7.48
7.09
–
7.50
7.12
–
7.51
7.52
After exposure to light
Difference (x4)
Before exposure to light
7.0
7.5
Figure 3. Comparison of the 1H-NMR spectra of ‘snail’ dye reduced in D2O
before and after exposure to light. The difference shows two signals at 7.12
and 7.51 ppm consistent with the disappearance of bromoindigo.
Before contact
with wool
After contact
with wool
7.8
7.6
7.4
7.2
7.0
Figure 2. Comparison between the 1H-NMR spectra of ‘snail’ dye reduced
in D2O before and after contact with wool. The difference spectrum shows
four signals at 7.11, 7.18, 7.40 and 7.64 that are assigned to indigo.
chemical shifts of the pure compounds (Table 1) thatwe measured
and found to be consistent with previously reported values.[25]
A comparison of the spectra (Fig. 2) before and after the
exposure to the wool shows clearly the removal of the indigo
signals at 7.11, 7.18, 7.49 and 7.64. The chemical shifts are sensitive
to concentration and pH and are therefore slightly different than
those of the pure compounds. Two signals at 7.41 and 7.78 ppm
increase upon exposure to wool and represent a compound that
leaches out of the wool. These signals could be used during the
dyeing process to determine how much of the dye has been
absorbed. They also provide a potential tool for kinetic studies on
the absorption of the dye.
Figure 4. DOSY spectrum showing the difference between protein and
leucodye.TheDOSYspectrumofpureleucoindigoisaddedforcomparison.
There is also a discernable difference before and after exposure
to light with signals corresponding to bromoindigo at 7.12 and
7.51 ppmdisappearing(Fig. 3).Thisprovesthatthedebromination
takes place after reduction and before the solution is diluted with
(NH4)2SO4 or brought into contact with the wool.
indigo. TheMurextrunculusdyecontainsmostlyproteinsowetried
to determine if the protein has any role in the process. The fact
that the wool selectively extracts the dye and not the protein from
solutiondoesnotindicatethattheproteinplaysanyrole.TheDOSY
spectrum (Fig. 4) of the leuco dye shows the dye diffusing much
faster than the protein although not as fast as a pure leucoindigo
solution. This indicates that the dye is not strongly attached to the
The question arises as to whether there is a scientific basis for
the religious requirement to use the ‘snail’ dye rather than plant
c
wileyonlinelibrary.com/journal/mrc
Copyright ꢀ 2010 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2010, 48, 892–895

NMR spectroscopic study of the Murex trunculus dyeing process
protein although the presence of protein increases the viscosity of
the solution. If there is any scientific justification for the religious
requirement for using ‘snail’ dye then it must come from the small
amount of indirubin or residual 6-monobromoindigo that may
have some effect on the color.
[3] Midrash Tanchumin, Numbers, Shlach, c. 850 CE.
[4] M. Borstein, The Tekhelet, Sifriati: Jerusalem, 1990.
[5] I. Hertzog, TheRoyalPurple and the BiblicalBlue, PhD Thesis, London
Univ, 1911, reprinted in 1987 by A. Shpneir.
[6] I. I. Ziderman, J. Soc. Dyers Colour. 1981, 97, 362.
[7] O. Elsner, E. Spanier, The dyeing with Murex extracts. An unusual
dyeing method of wool to the biblical sky blue, 7th International
Wool Textile Research Conference, Tokyo, Japan, Sept. 1985.
[8] I. I. Ziderman, Rev. Prog. Coloration 1986, 16, 46.
[9] O. Elsner, Dyes Hist. Archaeo. 1991, 10, 1.
Conclusions
[10] A. N. Padden, V. M. Dillon, P. John, J. Edmonds, M. D. Collins, Nature
NMR spectroscopy can be used as a non-invasive tool to follow the
debromination and absorption of leucoindigos on wool. Further
proof has been provided that the light catalyzed debromination
stage takes place completely in the leucodye and prior to contact
with the wool. The selective disappearance of leuco dye from
the solution spectrum after contact with wool shows that the dye
already forms a strong attachment with the wool prior to oxidation
and that it is not the oxidation that initially binds the dye to the
wool. There is no evidence that the protein, which makes up the
bulk of the commercial dye, plays any role in the dyeing process.
Following the chemical processes during the leuco stage, opens
up the possibility of correct problems that occur during dyeing
before it is too late thereby increasing reliability.
1998, 396, 225.
[11] A. N. Padden, V. M. Dillon, J. Edmonds, M. D. Collins, N. Alvarez,
P. John, Int. J. System. Bacter. 1999, 49, 1025.
[12] A. N. Padden, P. John, M. D. Collings, R. Hutson, A. R. Hall, J.Archaeo.
Sci. 2000, 27, 953.
[13] Z. C. Koren, Dyes. Hist. Archaeo. 2005, 20, 136.
[14] H. Fouquet, H.-J. Bielig, Angew. Chim. Int. Ed. 1971, 10, 816.
[15] I. I. Ziderman, Chem. Brit. 1986, 22, 419.
[16] L. A. Driessen, Melliand Textilberichte 1944, 26, 66.
[17] J. Van Alphen, Rec. Trans Chim. Pays-Bas 1944, 63, 95.
[18] Vitruvius, ‘‘De Architectura’’ Libra VII, Ch. 13. Rome, c. 25 BCE.
[19] B. Sterman, B’Or Hatorah 2001, XI, 185.
[20] C. J. Cooksey, Molecules 2001, 6, 736.
[21] Z. C. Koren, Michrochim. Acta. 2008, 162, 381.
[22] Z. C. Koren, J. Soc. Dyers Colour. 1994, 110, 273.
[23] I. I. Ziderman, Dyes Hist. Archaeo. 2001, 16/17, 87.
[24] I. I. Ziderman, Bathochromic Effect of Heat on 6-Bromoindigo 22nd
Annual Meeting on Dyes in History and Archaeology, Riggisberg,
Switzerland, Oct. 2003.
References
[25] G. Voss, J. Soc. Dyers Colour. 2000, 116, 87.
[1] Talmud, Tractate Menachot 44a, Babylon c. 500 CE.
[2] Talmud, Tractate Brachot 61b, Babylon c. 500 CE.
c
Magn. Reson. Chem. 2010, 48, 892–895
Copyright ꢀ 2010 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/mrc