Spectrochimica Acta Part A 61 (2005) 1395–1401
Non-destructive Raman analyses – polyacetylenes in plants
Bernhard Schrader a, ∗ , Hartwig Schulz b , Malgorzata Baranska b, c , George N. Andreev d ,
Caroline Lehner e , Juergen Sawatzki e
b
a Institut für Physikalische und Theoretische Chemie, Universität Duisburg-Essen, Soniusweg 20, D-45259 Essen, Germany
Federal Centre for Breeding Research on Cultivated Plants, Institute for Plant Analysis, Neuer Weg 22-23, D-06484 Quedlinburg, Germany
c Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland
d Department of Chemistry, University of Plovdiv, Bulgaria
e Bruker Optik GmbH, Rudolf-Planck-Strasse 27, D-76275 Ettlingen, Germany
Received 22 September 2004; received in revised form 15 October 2004; accepted 15 October 2004
Dedicated to Professor James Durig thanking for more than 30 years of friendship, personal and scientific contacts.
Abstract
Ferdinand Bohlmann has described the isolation, the identification and the structure elucidation of acetylene compounds in many plants,
and confirmed it by its synthesis. We have recorded the Raman spectra of most of these plants non-destructively by FT–Raman spectroscopy
using radiation at 1064 nm. We could not observe any interfering fluorescence. We found acetylene compounds in some plants, even distinct
compounds with different concentration in various parts of it. The distribution of the different compounds over the plant can be observed and
their changes during the ontogenesis can be followed by a FT–Raman mapping technique. Of special help is a library of Raman and IR spectra
and the structure of the compounds, synthesized by Bohlmann. Thus, the Raman technique allows analyses in a very short time replacing the
usual time-consuming separation procedures and avoiding artefacts during clean-up procedures.
© 2004 Elsevier B.V. All rights reserved.
Keywords: Non-destructive analyses by NIR–FT–Raman spectroscopy; Distribution of natural compounds in plants; Library of IR and Raman spectra of natural
compounds of plants; Application in breeding; Cultivation and quality control
1. Introduction
By exploring the most promising applications of
FT–Raman spectroscopy with excitation by a Nd:YAG
laser at 1064 nm we found that this technique allows nondestructive analyses of works of art, of animal and plant tissues [1,2] perfectly. Particularly, the disturbing fluorescence
of the enzymes and coenzymes of all cells, especially the
photosynthetic machinery in plants, produced mostly by all
other Raman techniques using excitation with visible light
and even at 785 and 830 nm, is avoided. The Raman spectra
∗
Corresponding author. Tel.: +49 201 460638; fax: +49 201 466650.
E-mail addresses: [email protected] (B. Schrader),
[email protected] (H. Schulz), [email protected] (M. Baranska),
[email protected] (G.N. Andreev), [email protected]
(C. Lehner).
1386-1425/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2004.10.048
of various terpenes in plants had been recorded already [3,4].
We were especially surprised to see strong Raman bands at
about 2230 cm−1 in the Raman spectra of several plants. Of
course, these bands give a proof for acetylenic bonds.
Raman spectra of acetylene and di-acetylene have been
published already in 1935 [5,6]. Acetylene shows a Raman
band at 1973 cm−1 , diacetylene (having a center of symmetry) produces a Raman band at 2183 and an IR band at
2085 cm−1 of the (C≡C)2 -in-phase- and out-of-phase vibrations, respectively. The first Raman analysis of an acetylene
compound in a plant, the carlinaoxide, has been published
already in 1935 [7].
Unsubstituted polyacetylenes are extremely unstable and are known to explode violently. Nevertheless,
Else Kloster–Jensen succeeded in preparing triacetylene,
tetraacetylene and even pentaacetylene and in recording their
Infrared, UV and NMR spectra [8]. Their UV spectra and
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B. Schrader et al. / Spectrochimica Acta Part A 61 (2005) 1395–1401
Fig. 1. FT–Raman spectrum of below: the bottom of the flower heads of Erigeron neclectus; above: compound I [16] lachnophyllum lactone. The drawing
copied into the figure is taken from [23].
their photoelectron spectra were discussed in detail [9]. The
Raman spectrum of triacetylene allowed the calculation of its
force field [10].
Baranovic and coworkers discussed the spectra of
oligo(phenyldiacetylenes) [11]. Furthermore, Raman and infrared spectra of various acetylenes have been discussed in
[12] and are published in the Raman/IR Atlas [13].
We were especially attracted by the work of Ferdinand
Bohlmann. He has found polyacetylenes in many plants, especially in species of Umbelliferae and Compositae families already since about 1955. Bohlmann has been by a
wide margin the most productive of all chemists dealing
with natural substances in higher plants. Winterfeldt reviewed Bohlmann’s life’s work and gives the list of his 1453
publications [14], published during the years 1948–1992.
Bohlmann’s early work concerning acetylenes in plants until 1963 have been summed up by Bohlmann and Sucrow in
the paper: ‘Natürlich vorkommende Acetylenverbindungen’
[15]. We sent a list of 57 botanical names given in this paper
to several Botanical Gardens in Germany, asking for samples
of living plants with this name. We investigated the Raman
spectra of many of them, especially of the flowers, leaves and
roots separately and found weak or strong bands in the range
of about 2000 cm−1 in several samples. The most important
of these measurement results are described in the present paper.
2. Experimental
The Raman spectra presented in this paper were
mainly recorded using an instrument equivalent to the
NIR–FT–Raman Spectrometer BRUKER RFS 100 with a
diode-pumped Nd:YAG laser, emitting maximal 350 mW at
1064 nm and a Germanium detector, cooled with liquid nitrogen. Most spectra have been recorded using a sample arrangement shown in Fig. 1c and f of [1] with a resolution of
4 cm−1 using a laser power of 150 mW supplied by an unfocused laser beam and with a recording time of 15–30 min.
Two-dimensional mappings were performed using an xy
stage, a mirror objective, a prism slide and a suitable software
to control the xy stage and the three-dimensional data processing. Raman mapping of the inflorescence of Bidens ferulifolia was performed from above over an area of 9 mm × 9 mm
with a spatial resolution of 250 ␮m and from the side over an
area of 7.2 mm × 10 mm with a spatial resolution of 200 ␮m
(Fig. 8). The sample was irradiated with a focused laser beam
of 100 mW with a diameter of about 0.1 mm. With a spectral resolution of 4 cm−1 , eight or six scans respectively were
collected at each measured point.
3. Results and discussion
The concentration of polyacetylenes in plants is in the order of 0.01–1%. Bohlmann studied the natural compounds in
plants usually by chopping the whole plants, extracting them
with a petrolether/diethylether mixture and sending the solution via a small chromatographic column through a sample
cell of an UV spectrometer. Polyacetylenes are shown up by
strong bands, due to the system of triple and double bonds in
the molecule [14]. This allowed him to optimise the extraction procedure and to prepare pure products. Bohlmann elucidated their structure by interpreting these bands and combining this with the results given by the other instrumental techniques of that time. Usually he confirmed it by preparation of
the corresponding synthetic substances. Polyacetylenes are
very sensitive, especially against heat and light, they some-
B. Schrader et al. / Spectrochimica Acta Part A 61 (2005) 1395–1401
times explode during distillation or by determination of the
melting point. They are best stored in the dark and under
nitrogen.
The function of polyacetylenes in plants is not yet clear.
Some of them are very poisonous, and phototoxic. Polyacetylenes in fungi may show antibiotic properties [15].
In the Bohlmann/Sucrow paper [15] the compounds are
ordered by the structural features, especially the number of
C≡C and C C bonds and their substituents. The formulae
are arranged together with the names of the compounds, the
names of the plants from which they were extracted and the
citation of the publication, describing the isolation, the analyses and its syntheses.
We investigated many plants described in the publication
[15] by FT–Raman spectroscopy and found the bands of polyacetylenes in some of them. We noticed that they are not uniformly distributed over the whole plant, but concentrated on
specific parts of the plant—blossoms, leaves, roots, or seeds.
We even found distinctive compounds in different parts of
the same plant.
Fortunately, a library of the infrared and Raman spectra
obtained from 1505 samples of the Bohlmann collection of
natural compounds, which have been stable enough to survive
the storage, is now available [16]. These spectra are combined
with the molecular structures, given by the individual molfiles. They include spectra of 43 mono-, di- and triacetylenes,
further of alkaloids, coumarins and many other rarely known
secondary metabolites.
Of the 43 Raman spectra with acetylene groups from the
library [16] we related the observed bands in the region about
2200 cm−1 to the molecular structure. In the library, there are
14 molecules with one C≡C bond, 23 with (C≡C)2 and 6 with
(C≡C)3 groups. All acetylene groups are disubstituted. It is
surprising that most spectra of the molecules with mono- and
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diacetylene groups have a very strong band at 2230 cm−1 ,
especially if the adjoining group(s) of the triple bond(s) are
aliphatic. Some of the molecules show a weak satellite band
at about 2190 cm−1 , which may be due to the 13 C isotopic
substitution of the triple bonds [17]. Satellite bands at about
2290 cm−1 were attributed to Fermi resonance [12]. Only in
the group of the six triine-molecules, the strongest bands are
at lower frequencies: 2190–2118 cm−1 . It is interesting that
also the compounds with octadehydrodibenzo[12]annulene
and dodekadehydrotribenzo[18]annulene [12] showed bands
at 2191 and 2198 cm−1 , respectively.
The Raman spectrum of the unsubstituted triacetylene
showed two very strong bands, at 2212 and 2019 cm−1 , due
to the in-phase stretching vibration of all three triple bonds
and the out-of-phase vibration of the same bonds [10].
In the bottom of the flower of Erigeron neclectus we found
a strong band at 2198 cm−1 (Fig. 1). According to Bohlmann
it can be attributed to the lachnophyllumlactone, I in [15].
We could not find a spectrum of exactly this compound in
the library, however only one with a further double bond in
the side chain. This explains its lower frequency (2178 cm−1 )
compared to that in the plant (2198 cm−1 ).
Coreopsis grandiflora (Fig. 2) shows in the roots a band at
2194 cm−1 . According to Bohlmann [14] the isolated compound V is a monoacetylene with a substitution by a thiophene ring. We did not find this compound in [16], however an example with one thiophene ring substituted by
monoacetylenes has a similar spectrum.
Measurements of the flowers of the ox-eye daisy
(Chrysanthemum leucanthemum) show the FT–Raman spectrum presented in Fig. 3; it can be seen there that the isolated
trans-dehydromatricariaester (LII in [15]), of which the spectrum is shown above exactly coincides with the spectrum of
this flower.
Fig. 2. FT–Raman spectrum of below: the roots of Coreopsis grandiflora; above: reference spectrum of a thiophene substituted monoacetylene (compound V
[16]).
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B. Schrader et al. / Spectrochimica Acta Part A 61 (2005) 1395–1401
Fig. 3. FT–Raman spectrum of below: the blossoms of Chrysanthemum leucanthemum; above: compound LII [16] trans dehydromatricariaester. The drawing
copied into the figure is taken from [23].
The blossoms of cornflowers (Centaurea cyanus) show
the spectrum of Fig. 4 which is in good accordance with
the spectrum of LIV in [15]. The intensity of both ν(C≡C)
bands are reverse which may point out that another acetylene
compound or other isomer is also present in the blossoms.
The flower heads of Centaurea ruthenica and the leaves of
Carthamus lanatus show polyacetylene bands of which the
position (2166.7 and 481.4) is nearly identical with that of
LXII in [15] (Fig. 5).
When exposed to abiotic stress during harvesting, transportation, storage and processing, carrots are able to produce
a bitter off-flavour. Recently, it has been found, that several diacetylenes such as falcarinol and falcarindiol contribute to the
bitter taste of carrots [18]. Although these substances were
already identified in various Apiaceae species before, their
sensoric properties were yet not known [19]. Fig. 6 shows a
spectrum of a substance of the library, which shows a similar spectrum as the natural diacetylenes occurring in carrots.
The intensity of the bands, due to the carotene at 1520 and
1156 cm−1 are the strongest in the spectrum.
Fig. 7 shows the spectrum of the leaf and the flower of
apache beggarticks (Bidens ferulifolia, family Compositae).
This species, native to Guatemala and Mexico, is exclusively
used as horticultural plant for hanging baskets and window
boxes. Other species of the genus Bidens are widely used in
Chinese medicine, such as Bidens pilosa and Bidens campy-
Fig. 4. FT–Raman spectrum of below: the heads of Centaurea cyanus (cornflower); above: compound LIV [16]. The drawing copied into the figure is taken
from [23].
B. Schrader et al. / Spectrochimica Acta Part A 61 (2005) 1395–1401
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Fig. 5. FT–Raman spectrum of line a: bottom of the flowers of Centaurea ruthenica; line b: leaves of Carthamus lanatus; above: compound LXII [16]. The
drawing copied into the figure is taken from [23].
lotheca. Previous examinations of these species resulted in
the identification of several lipophilic polyacetylenes and
polyacetylene glucosides [20,21] which seem to be mainly
responsible for the described medicinal properties. Chang et
al. [22] described one of the acetylene compounds, occurring
in B. pilosa, as a phenyl substituted triine. We found that the
reference spectrum of this substance is similar to that obtained from the flowers of B. ferulifolia (Fig. 7). In addition
both carotene bands at 1527 and 1158 cm−1 are very intense.
Contrary to that, the Raman spectrum of the leaves show
a polyacetylene signal at lower wavenumbers (2135 cm−1 ).
More detailed work, including HPLC-MS identification of
the individual unknown polyacetylenes in B. ferulifolia is
necessary to allow a more precise interpretation of these spectra.
The Raman map of the inflorescence of B. ferulifolia gives
another unexpected information: The upper row in Fig. 8
shows Raman maps and the picture from above—the left map
shows the distribution of carotenes, which occur obviously in
the petals, whereas the polyacetylenes (middle maps) occur
mainly in the stamen, this is also shown in the middle map of
the lower row in Fig. 8 showing a greater enlargement of the
flower from aside. The presented data correspond very well
with previously published results reporting the polyacetylene
distribution in the chamomile inflorescence based on the Raman mapping technique [4].
Fig. 6. FT–Raman spectrum of below: carrot root containing higher amounts of polyacetylenes; above: reference spectrum of a C18 diine.
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B. Schrader et al. / Spectrochimica Acta Part A 61 (2005) 1395–1401
Fig. 7. FT–Raman spectrum of line a: the flower; line b: the leaf of Bidens ferulifolia; line c: reference spectrum of a phenyl-substituted triine (compound L
[16]. The drawing copied into the figure is taken from [23].
Fig. 8. Upper row: maps and photo of the flower of Bidens ferulifolia from above, left map: distribution of carotenes, middle map: distribution of polyacetylenes;
lower row: middle and right: map of the polyacetylenes and photo of the flower from aside. The colours of the map describe the integral Raman intensity.
B. Schrader et al. / Spectrochimica Acta Part A 61 (2005) 1395–1401
In discussing the figures one has to take into account, on
the one hand that the library [16] contains only the spectra of
pure samples which were stable enough to be synthesized by
Bohlmann and which have survived the storage. On the other
hand, we recorded FT–Raman spectra of the plant where the
polyacetylenes are present mostly at low concentration, together with other substances in the biologic machinery of the
plant. It is therefore surprising how good the agreement of
the important bands of some samples – in the library versus
that in the plant – is!
We regard this as a proof of the importance of plant analyses by FT–Raman spectroscopy.
4. Conclusion
The non-destructive analysis of natural substances in
plants is of high importance in biochemistry, for exploring
sources of medicinal drugs and of raw materials for the pharmaceutical industry. This can be performed using FT–Raman
spectroscopy with excitation at 1064 nm and the sample arrangement described in Fig. 1c and f of [1].
Concentration and distribution of the natural compounds
in plants is dependent on the genotype and their taxonomy.
Raman spectroscopy helps the breeders to select high-quality
crossing progenies. Furthermore, this spectroscopic technique allows to monitor simultaneously the concentration
changes of various plant substances during ontogenesis and
based on this data to predict the optimal harvesting time.
Of great practical importance is the technique of mapping
of the Raman spectra over the whole plant or larger parts of
it. It shows the distribution of the compounds of interest and
which part of the plants contains their highest concentration.
The Raman technique allows quantitative analyses in a
very short time replacing the usual time-consuming separation procedures. Artefacts during clean-up procedures are
avoided.
Acknowledgements
We thank the Botanical Gardens of the Universities
Bochum, Dresden and Hohenheim for the supply of the plants
investigated during the preparation of this article. Furthermore the financial support of the Deutsche Forschungsgemeinschaft (DFG) in Bonn, Germany (grant number Schu
1401
577/7-1) is gratefully acknowledged. My wife Christa helped
me (BS) collecting the plants and to run and analyse their Raman spectra.
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