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Using Spectral Measurements to Differentiate Between Anthocyanin

and Betacyanin Containing Plant Species


Reisha D. Peters1*, Scott D. Noble2
1,2

Department of Chemical and Biological Engineering, College of Engineering, University of


Saskatchewan, 57 Campus Drive Saskatoon SK, S7N 5A9 Canada
*Corresponding Author. E-mail: reisha.peters@usask.ca

Abstract
Anthocyanins and betacyanins are classes of red-purple plant pigments that may be useful in
plant identification and health monitoring. Pure samples of anthocyanin and betacyanin were
obtained by extracting and isolating pigments found in red cabbage and beets. The resulting
samples were then used to identify the absorption spectra of both of these pigments. The
isolation process was carried out using thin-layer chromatography and column
chromatography through which sufficiently pure samples of anthocyanin and betacyanin
were obtained from red cabbage and beets respectively. These samples were analyzed by
measuring the reflectance from the thin-layer chromatography plates and the absorption of
the liquid-phase pigment extracts, resulting in spectral data for each pigment from 200 to
1400nm at 1-nm intervals.
Key words: anthocyanin, betacyanin, spectrophotometry, PROSPECT
1. Introduction
Anthocyanin and betacyanin are mutually exclusive red pigments found in plants
(Brockington et al., 2011). These pigments display slightly different spectral characteristics,
especially in the ultraviolet range where anthocyanin strongly absorbs light. Currently, highresolution spectral data for these pigments are not available or have not been obtained in the
range required for future study in distinguishing these pigments in vivo using modeled and
measured approaches. Future study requires data collected between 200 and 1400nm in 1nm intervals. This study was initiated to determine the spectrum of these pigments in this
range and resolution so that they could be applied to the PROSPECT leaf reflectance model
(Feret et al., 2008) in order to model differentiating spectral features between plants
containing opposing red pigments.
Anthocyanin is formed through a number of enzymatic pathways all originating from the
amino acid phenylalanine (Brockington et al., 2011). Within the anthocyanin family, there are
a number of alternate forms of the pigment which are categorized based on the side chains
attached. Many of these side chains are pH sensitive and the absorption spectra can change
based on the acidity level (Lee et al., 2005). In the case of red cabbage, the observable color
of the anthocyanin solution will change based on the acidity level, and will therefore shift the
absorption spectra. Consequently, it is important to maintain a pH similar to that which would
be present in the plant to avoid this absorption spectra shift. This was achieved by extracting
the anthocyanin in a 1% HCl in methanol solution which maintains the anthocyanin peak at
530nm (Siegelman & Hendricks, 1958). Cyanidin-3-glucoside was used as the reference in
the calculation of anthocyanin concentration as this is a prominent anthocyanin found in
many plants (Lee et al., 2005).
RodriguezSaona and Wrolstad (2001) performed anthocyanin purification using a C18
cartridge. First, the cartridge was prepared using two column volumes of methanol. Next the
anthocyanin sample was added to the cartridge. A solution of 0.01% HCl in water was used
to elute sugars and acids after which ethyl acetate was used to remove the phenolics.
Finally, the anthocyanins were eluted using 0.01% HCl in methanol.

Betacyanin is formed through enzymatic pathways originating from the amino acid tyrosine.
A side reaction uses additional amino acids in the formation of betaxanthin (Brockington et
al., 2011). Because betaxanthin is formed from an intermediate product of betacyanin
synthesis, it is found in almost all plants that contain betacyanin.
One method used to separate betacyanin from betaxanthin required two solutions to develop
the thin layer chromatography (TLC) plate (Bilyk, 1981). The first solution contained a 6:7:6:1
mixture of isopropanol:ethanol:distilled water:acetic acid and was used to develop the TLC
plate to 10 cm. The second solution was a 11:4:4:1 mixture of isopropanol:ethanol:distilled
water:acetic acid and was used to develop the same TLC plate to 15 cm. The original
experiment using this method was performed using a cellulose plate.
The fact that these two pigments are mutually exclusive is of particular interest in plant
identification. If one of these pigments is observed in the plant to be identified, all species
containing the other pigment can be eliminated (Noble, 2006). The degree to which this is
possible for in-situ spectral measurements of plant tissues is currently unknown. Modeling
the leaf reflectance by using a tool such as the PROSPECT model will be useful in identifying
detection limits and methods prior to assessment with actual plant reflectance data.
The PROSPECT model synthesizes leaf spectra by using a few input parameters. The
current version of the PROSPECT model provides three pigment specific absorption spectra
including chlorophyll a and b, carotenoids, and brown pigments (tannins) (Feret et al., 2008).
The model also uses the specific absorption spectra of water and dry matter as well as the
refractive index of leaf material. Finally, a structure parameter, N, allows for the thickness of
the leaf to be taken into consideration. Currently, the spectral data in the model exists in the
range of 400 to 2500 nm with 1-nm resolution. In order to be compatible with the model, it is
necessary that the new data also be in this form. Adding in the new betacyanin and
anthocyanin spectra will improve the model and allow for more accurate analysis and plant
identification.
2. Materials and methods
Extractions of the anthocyanin and betacyanin pigments were performed by extracting juices
from red cabbage (Brassica oleracea sp.) and beets (Beta vulgaris L. sp.), respectively, and
then using column chromatography to obtain individual pigments. These pure extracts were
then analyzed to determine their spectral identities and compared to existing data in the
literature.
2.1 Thin-layer chromatography testing
The first set of tests was performed using TLC plates (silica gel 60, 200 micron) and different
mobile phases to determine which combination provided the best separation and measure
the reflectance of each pigment. These tests were more beneficial for the beet (betacyanin)
tests because a similar type of silica gel was used on both the plate and in the column
whereas the cabbage (anthocyanin) was separated using a 300 mg C18 cartridge. A small
quantity of one pigment was applied several times to a plate and allowed to air dry. Once the
sample was dry, the plates were developed at an incline in a beaker with a few milliliters of
the mobile phase in the bottom of the beaker. Each plate was allowed to develop until the
mobile phase had run approximately 8 cm up the plate.
Acidified methanol was used as the mobile phase in the red cabbage separation. For the
beet sample, three different methods were tested as possible separation techniques. The
first method was carried out as described by Bilyk (1981), the second method used methanol
as the mobile phase and the third used distilled water.
2.2 Anthocyanin extraction
The anthocyanin pigments were extracted by macerating red cabbage in water and filtering
the mixture. To remove impurities and obtain a sample of pure anthocyanin the extract was

separated using column chromatography. A 300 mg C18 cartridge was used and the column
was prepared using the procedure outlined by RodriguezSaona and Wrolstad (2001) except
1% HCl in methanol was used as per Siegelman and Hendricks (1958). Once the
anthocyanin sample was eluted, it was analyzed immediately so that the pigment would not
degrade. This was done by diluting the sample and taking the spectral measurement.
2.3 Column packing
The chromatography columns used in the beet extraction were made using a small glass
pipette (interior diameter approximately 5.5 mm). First, a small cotton ball was wedged into
the bottom portion of the pipette (the part which begins to taper to a point) to prevent the
column contents from washing out of the bottom. A 5 mm layer of washed sea sand was
added on top of the cotton. The pipette was then filled to about two and a half centimeters
from the top with the stationary phase (silica gel 60, 230-400 mesh). Finally, another half
centimeter layer of sand was added above the silica gel in order to filter out any particles
such as plant matter. It was very important to ensure each layer was level as an uneven
surface would affect separation results.
2.4 Betacyanin extraction
The betacyanin and betaxanthin pigments were extracted by macerating red beet in water
and filtering the mixture. The resulting sample contained a large amount of both betacyanin
and betaxanthin, so further purification was performed with hopes of isolating these two
pigments. Two columns were prepared using silica gel; the first column used methanol as the
mobile phase and the second used water. The two-solution method described by Bilyk
(1981) was not used in the column extraction as the results from the TLC plates showed poor
separation between betacyanin and betaxanthin. The column preparation method is
described in section 2.3.
Two column-volumes of the mobile phase were used to prepare each column before the beet
juice was added. Once the beet juice had completely absorbed into the surface of the
column, water or methanol was added to keep the top portion of the column full. The beet
juice was allowed to flow through the column and the red (betacyanin) and yellow
(betaxanthin) fractions were collected separately.
2.5 Spectral measurements
Spectral measurements were taken from all thin-layer chromatography plates using an
Ocean Optics Maya 2000PRO spectrometer (Ocean Optics, Dunedin, FL, USA). These
measurements were used to identify the general reflectance of each pigment which was
helpful in identifying the absorption peak and provided data for comparison against the
solution measurements. These spectra were obtained between 200 and 1120 nm at 0.475
nm intervals.
All spectral measurements of solutions were taken using a Cary 5G UV-visible-NIR
spectrophotometer (Agilent Technologies, Mississauga, Can.) and were sampled at 1-nm
intervals between 200nm and 1400nm. Blank solutions of the mobile phase were used as
zero references for each spectrum.
3. Results and discussion
The thin-layer chromatography plate separation for anthocyanin is displayed in Fig. 1a; a
vibrant purple anthocyanin band can be observed near the top of the plate. In the beet
sample tests, the first method (using two solutions and developing the plate twice) did not
show very good separation. This lack of separation is likely due to the use of a silica gel plate
instead of a cellulose plate and so it was not used in the column chromatography. In Fig. 1,
the separations using water and methanol can be seen. Both mobile phases showed some
separation between the yellow and red pigments (betaxanthin and betacyanin) but the water
separation (Fig. 1c) was much greater. Fig. 1c shows a distinct separation between the bright

pink betacyanin pigment near the top and the dim yellow bettaxanthin pigment. The
e yellow
nk likely because the concentratio
c
on of betaxa
anthin is
band is much less prominent than the pin
an that of be
etacyanin.
less tha

FIG
GURE 1: Separation of pigments on
o TLC plate
es. (a) anthocyanin fro
om red cabb
bage
(acidifie
ed methano
ol mobile ph
hase) (b) be
etacyanin an
nd betaxantthin from be
eet juice (methanol
mobiile phase) and
a (c) beta
acyanin and betaxanthin from beett juice (wate
er mobile ph
hase)
nts from each
The refflectance measureme
m
e
band on the TL
LC plates were
w
converted to
absorpttion using eq
quation 1.

1
100
Abs = ln

%R

(1)

A 15-po
oint moving
g average was
w used to display the
e trend of ea
ach molar extinction
e
co
oefficient
spectrum and thes
se results ccan be seen in Fig. 2.. The antho
ocyanin reflectance wa
as taken
e darkest purple band at the top of
o the plate. The beet juice plate which was run with
from the
water as
a the mobile phasse was us
sed to ob
btain the betacyanin and beta
axanthin
measurrements fro
om the mo
ost intense pink and yellow are
eas of the plate resp
pectively.
Measurrements fro
om the beet juice platte run with methanol as the mobile phase are not
shown d
due to poorr separation of pigmentts.

and
FIG
GURE 2: Sp
pectral data obtained frrom TLC pla
ates for anth
hocyanin, betacyanin,
b
betaxanthin
n
Reason
nably pure samples
s
off anthocyan
nin and bettacyanin we
ere obtaine
ed from the column
chromatography separations. Betaxanth
hin was also extracted
d but the sample conttained a

conside
erable amou
unt of betaccyanin as we
ell. Because
e pure beta
axanthin cou
uld not be obtained,
o
the beta
acyanin wass subtracted
d out of the
e contamina
ated spectru
um. This wa
as accomplished by
scaling the pure betacyanin spectrum for zero betaxanthin
b
n absorption at 536 nm and
m from the contaminated one. Th
he molar extinction co
oefficient
subtractting the pure spectrum
spectra for the anth
hocyanin, betacyanin,
b
anthin are sh
hown in Fig
g. 3.
and betaxa

FIGUR
RE 3: Molarr extinction data for anthocyanin, betacyanin, and betaxanthin, calcculated
from
m liquid sam
mples
The mo
olar extincttion coefficcient spectrum for ea
ach pigment was calculated ussing the
spectrum peak and
d the specific molar exttinction coe
efficient at th
hat peak. Fo
or anthocya
anin, this
% HCl in methanol
m
(Siegelman & Hendrickss, 1958).
was 34,300 Lmol-11cm-1 at 530 nm in 1%
According to von Elbe (2001
1), betacya
anin should have a peak at 538
8 nm with a molar
on coefficie
ent of 38,000 Lmol-11cm-1. Fina
ally, betaxa
anthin shou
uld have a molar
extinctio
extinctio
on coefficient of 48,000
0 Lmol-1cm-1 at 480 nm
m (Ganda-H
Herrero et al., 2005).
The re
esults from
m the TLC
C plates and
a
the co
olumn extrracts displayed very similar
characte
eristics sho
owing the sa
ame absorp
ption peakss at 535 and
d 536 nm fo
or anthocya
anin and
betacya
anin, respec
ctively. The
e betaxanth
hin spectrum
m from botth measure
ements had a peak
around 475 nm. A noticeable difference between
b
the
e two measurement me
ethods is th
he shape
h spectrum in the UV
V range (be
elow 300 nm). While the absorp
ption in the column
of each
extractio
ons appearrs to increasse as the wavelength
w
a
approachess 200 nm, the reflectan
nce data
would ssuggest tha
at there is m
more than just comple
ete absorption in this range.
r
This is most
likely due to the method by which eacch measure
ement was taken: the
e TLC plate
es were
ed by mea
asuring the
e reflectancce and converting th
his value to
t an apprroximate
analyze
absorpttion while th
he results ffrom the co
olumn chrom
matographyy were obta
ained by me
easuring
t
the sample.
the transmittance through
T
plates directly
d
provvides the sp
pectrum of a pure beta
axanthin
The datta obtained from the TLC
sample but the data obtained
d from the liquid samples does no
ot. For this reason, the
e results
e TLC plate
es would be
e a better op
ption for use in the currrent model. However, if better
from the
data co
ould be colle
ected using
g the colum
mn chromato
ography, th
hat data would be morre suited
for use in the model. This cou
uld be acco
omplished by
b extracting
g pigments from a yelllow beet
mple would have a lower
l
betacyanin con
ncentration, therefore making
cultivar.. This sam
contamination lesss of a proble
em.

4. Conclusion
The absorption spectrum of anthocyanin, betacyanin and betaxanthin were obtained with a
resolution of 1 nm in the 200-1400 nm range. These data were collected using two methods
and the two sets of results were consistent with one another above 400 nm. In the UV range
of the spectrum the data had very different characteristics; this is likely due to the collection
method used and not the actual spectra of the pigments.
Anthocyanin and betacyanin showed very similar characteristics in the visible range but
anthocyanin displayed much greater absorption in the UV range. This is likely a characteristic
which could be used to distinguish between these two pigments. Also, betaxanthin displayed
an absorption peak at 475 nm which would only be present in the case of betacyanin
production. This is another useful characteristic which would be helpful in distinguishing
opposing red pigments.
Using the new spectral data collected, the PROSPECT model will be modified to include
anthocyanin and betacyanin. The improved model will be tested using spectral reflectance
data from anthocyanin and betacyanin containing plants.
5. References
Bilyk A. (1981). Thin-layer chromatographic separation of beet pigments. Journal of Food
Science, 46, 298-299.
Brockington, S.F., Walker, R. H., Glover, B. J., Soltis, P. S., and Soltis, D. E. (2011).
Complex pigment evolution in the caryophyllales. New Phytologist, 190, (4): 854-864.
Feret, J. B., Francois, C., Asner, G. P., Gitelson, A. A., Martin, R. E., Bidel, L. P. R., Ustin, S.
L., le Maire, G., & Jacquemoud, S. (2008). PROSPECT-4 and 5: Advances in the leaf optical
properties model separating photosynthetic pigments. Remote Sensing of Environment, 112,
(6): 3030-3043.
Ganda-Herrero, F., Escribano, J., & Garca-Carmona, F. (2005). Betaxanthins as substrates
for tyrosinase. An approach to the role of tyrosinase in the biosynthetic pathway of betalains.
Plant Physiology, 138, 421-432.
Lee, J., Durst, R. W., & Wrostad, R. E. (2005). Determination of total monomeric anthocyanin
pigment content of fruit juices, beverages, natural colorants, and wines by the pH differential
method: Collaborative study. Journal of AOAC International, 88, 1269-1278.
Noble, S. D. (2006). Combining spectral and spatial information for automated plant
identification. Ph.D. Thesis, University of Guelph, Guelph, ON
RodriguezSaona, L. E., & Wrolstad, R. E. (2001). Extraction, isolation, and purification of
anthocyanins. Current Protocols in Food Analytical Chemistry, F1.1.1-F1.1.11, John Wiley &
Sons, Inc.
Siegelman, H. W., & Hendricks, S. B. (1958). Plant photocontrol of anthocyanin synthesis in
apple skin. Plant Physiology, 33, (3): 185-190.
von Elbe, J. H. (2001). Betalains. Current Protocols in Food Analytical Chemistry, F3.1.1F3.1.7, John Wiley & Sons, Inc.