Journal of Chromatography B, 941 (2013) 5461

Contents lists available at ScienceDirect

Journal of Chromatography B
j our nal homepage: www. el sevi er . com/ l ocat e/ chr omb
Versatile solvent systems for the separation of betalains from
processed Beta vulgaris L. juice using counter-current chromatography
Aneta Sprna-Kucab
a,
, Svetlana Ignatova
b
, Ian Garrard
b
, Sawomir Wybraniec
a
a
Department of Analytical Chemistry, Institute C-1, Faculty of Chemical Engineering and Technology, Cracow University of Technology, ul. Warszawska 24,
Cracow 31-155, Poland
b
Brunel Institute for Bioengineering, Brunel University, Uxbridge, Middlesex, United Kingdom
a r t i c l e i n f o
Article history:
Received 13 May 2013
Received in revised form
27 September 2013
Accepted 1 October 2013
Available online 10 October 2013
Keywords:
Betanin
Betalains
Betacyanins
Counter-current chromatography
Beta vulgaris L
a b s t r a c t
Two mixtures of decarboxylated and dehydrogenated betacyanins from processed red beet roots (Beta
vulgaris L.) juice were fractionated by high performance counter-current chromatography (HPCCC) pro-
ducing a range of isolated components. Mixture 1 contained mainly betacyanins, 14,15-dehydro-betanin
(neobetanin) and their decarboxylated derivatives while mixture 2 consisted of decarboxy- and dehydro-
betacyanins. The products of mixture 1 arose during thermal degradation of betanin/isobetanin in mild
conditions while the dehydro-betacyanins of mixture 2 appeared after longer heating of the juice from B.
vulgaris L. Two solvent systems were found to be effective for the HPCCC. A highly polar, high salt concen-
tration system of 1-PrOHACN(NH
4
)
2
SO
4
(satd. soln)water (v/v/v/v, 1:0.5:1.2:1) (tail-to-head mode)
enabled the purication of 2-decarboxy-betanin/-isobetanin, 2,17-bidecarboxy-betanin/-isobetanin and
neobetanin (all from mixture 1) plus 17-decarboxy-neobetanin, 2,15,17-tridecarboxy-2,3-dehydro-
neobetanin, 2-decarboxy-neobetanin and 2,15,17-tridecarboxy-neobetanin (from mixture 2). The other
solvent system included heptauorobutyric acid (HFBA) as ion-pair reagent and consisted of tert-butyl
methyl ether (TBME)1-BuOHACNwater (acidied with 0.7% HFBA) (2:2:1:5, v/v/v/v) (head-to-
tail mode). This system enabled the HPCCC purication of 2,17-bidecarboxy-betanin/-isobetanin and
neobetanin (from mixture 1) plus 2,15,17-tridecarboxy-2,3-dehydro-neobetanin, 2,17-bidecarboxy-2,3-
dehydro-neobetanin and 2,15,17-tridecarboxy-neobetanin (mixture 2). The results of this research are
crucial in nding effective isolation methods of betacyanins and their derivatives which are meaningful
compounds due their colorant properties and potential health benets regarding antioxidant and cancer
prevention. The pigments were detected by LC-DAD and LCMS/MS techniques.
2013 Elsevier B.V. All rights reserved.
1. Introduction
Counter-current chromatography (CCC) is a liquidliquid chro-
matography technique which was invented in the early 1960s
[1,2]. In high-speed counter-current chromatography (HSCCC),
high speed coil rotation around its own axis and a central axis
(planetary motion) generates a centrifugal eld to retain the liquid
stationary phase in the coil. The mobile phase is pushed through
Abbreviations: ACN, acetonitrile; BuOH, butanol; CCC, counter-current
chromatography; CID, collision induced dissociation; EtOH, ethanol; HFBA, hep-
tauorobutyric acid; HPCCC, high-performance counter-current chromatography;
HSCCC, high-speed counter-current chromatography; KD, partition coefcient;
MeOH, methanol; PFCA, peruorocarboxylic acid; RP-HPLC, reversed phase high-
performance liquid chromatography; PrOH, propanol; TBME, tert-butyl methyl
ether; TFA, triuoroacetic acid.

Corresponding author: Tel.: +48 12 628 30 74.

E-mail addresses: anetasporna@chemia.pk.edu.pl, anetasporna1@wp.pl
(A. Sprna-Kucab).
with a pump. The g-level produced is an effect from the coil rota-
tion and for a typical HSCCC machine, it is between 55 and 80
g-level [13]. High-performance counter-current chromatography
(HPCCC) is the name given to a high g-level machine (240g) and
was introduced by the Brunel Institute for Bioengineering [4].
The application of CCC to the fractionation and purication of
natural plant pigments has been shown in numerous publications
[511].
Beta vulgaris L. is increasingly utilized as a source of natural
food dyes due to a growing interest of consumers in its potential
health benets (antioxidant, anticarcinogenic) and the non-toxic
features of betalains. Since some synthetic pigments are considered
as toxic and harmful [12] there is a demand for natural equiva-
lents. Choosing a suitable solvent systemfor betalains purication
is challenging due to their low stability in some physicochemical
conditions [1214].
A fewpathways of betalain degradation and transformation are
known, such as decarboxylation, dehydrogenation, hydrolysis and
deglycosylation. Decarboxylation of betalains can occur at either
1570-0232/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jchromb.2013.10.001
A. Sprna-Kucab et al. / J. Chromatogr. B 941 (2013) 5461 55
Fig. 1. Steps of betanin and its diastereomer isobetanin thermal degradation path-
ways.
C-2, C-15 or C-17 carbon positions, however, usually occurs at C-2
and C-17. The dehydrogenation is observed at C-2,3 and C-14,15.
The products of betanindegradationare usually more stable, which
makes theminterestingmaterial for further applicationinthephar-
maceutical and food industries (Fig. 1) [12,1517].
Preparativeisolationof unstablebetalains byHPLCis oftenprob-
lematic due to the catalytic action of the solid stationary phase
causing pigment degradation, therefore, new separation methods
such as counter-current chromatography create an important pos-
sibility of obtaining pure pigments. CCC enables the use of different
stationary phases through the application of different solvent sys-
tems without the need to buy a new column. In addition, modern
CCC technology is as easy as HPLC to scale up to preparative and
pilot levels.
Hitherto, the rst successful isolation and purication of more
hydrophobic betalains by HSCCC was carried out in a solvent sys-
temconsisting of TBMEBuOHACNwater (acidiedwithion-pair
reagents TFA or HFBA) [69]. The addition of ion-pair reagents
results in a different chromatographic behavior of betalains e.g.
longer retention time of betalains in RP-HPLC [6,18]. The addition
of ion-pair additives to the CCC solvent systems changes the parti-
tion coefcient (K
D
) of betalains and efciently shifts them to the
organic phase, creating a new possibility for separation of these
highly polar plant pigments [6,8,18].
The novelty of this contribution is a fractionation and isolation
of decarboxylated and dehydrogenated derivatives of betanin from
processed B. vulgaris L. juice using HPCCC. These mixtures of beta-
lains have never been separated by CCC, which would be a useful
technique for the separation as its liquid stationary phase does not
catalyze degradation or cause irreversible adsorption and loss of
the components, in the way solid stationary phases may do. The
differences inelutionproles tracedinthe HPCCCandHPLCsepara-
tions were of special interest and were indicated by recent betalain
separations [6,8,18]. The HPCCC process was accomplished using
two different types of solvent systems: an ion-pair system and a
high salt concentration system. The high salt solvent systems were
used for the rst time in order to separate betalains. Whilst ion-
pair solvent systems have been reported before for the separation
of non-decarboxylatedandnon-dehydrogenatedbetalains, nothing
is known about their efciency in the separation of decarboxy-
lated and dehydrogenated betacyanins. Furthermore, the presence
of toxic ion-pair agents makes these systems less attractive for use
in the food industry [69].
2. Experimental
2.1. Reagents
HPLC-grade acetonitrile (ACN), 1-propanol (1-PrOH), ethanol
(EtOH), 1-butanol (1-BuOH), ammonium sulphate, tert-butyl
methyl ether (TBME), TFA and HFBA were obtained from Fisher
Chemicals (Loughborough, UK). Water was deionized (Purite,
Thames, Oxon, UK). HPLC-grade formic acid, methanol (MeOH)
were obtained fromPOCH (Gliwice, Poland).
2.2. The preparation of the crude pigment extracts
Two groups of betacyanins with different decarboxylation and
dehydrogenation levels were obtained by thermal treatment of B.
vulgaris L. juice and then analyzed by LC-DAD and LCMS/MS. The
juice was obtained from red beet roots (purchased as whole beet
roots fromthe local market, Krakw, Poland) which were washed,
hand-peeled, cut into small pieces andsqueezedina juice extractor
(Zelmer, Rzeszw, Poland) (Table 1).
The heating of betalain mixtures in the juice was performed
at 85

C for 30min (mixture 1) and 60min (mixture 2), both

acidied with 0.2% (v/v) formic acid according to a previous proce-
dure [12]. The mixtures were separately puried on a preparative
solid-phase extraction (SPE) column packed with C-18 reversed
phase material (Merck, Darmstadt, Germany) [12]. The eluates in
aqueous-acetonitrile solution were then concentrated by rotary
evaporator and then freeze-dried for the HPLC analysis and the
HPCCC experiments.
2.3. Apparatus
A semi-preparative Spectrum HPCCC J-type modern hydro-
dynamic CCC instrument was used (Dynamic Extractions, Slough,
UK) for the separation of betanin/isobetanin and their decarboxy-
and dehydro-derivatives (mixtures 1 and 2).
The Spectrum HPCCC had a maximum rotation speed of ca.
1600rpm (R=75mm, 240g eld). The instrument was equipped
with two columns of 143.5ml total capacity, 71mlong and 1.6mm
i.d. The mobile phase was pumped in the tail-to-head direction
(systemAI) and head-to-tail direction for systemBIV (Table 2).
The initial scouting runs were performed on the analytical size
Mini HPCCC instrument (systems AIAIII, and BIBIV) supplied by
Dynamic Extractions (Slough, UK). The Mini HPCCC was equipped
with a single 7cm diameter column made with 0.8mm i.d. poly-
tetrauorethylene (PTFE) tubing: 18.2ml capacity, column length
56 A. Sprna-Kucab et al. / J. Chromatogr. B 941 (2013) 5461
Table 1
Chromatographic, spectrophotometric and mass spectrometric data of the pigments identied in the crude mixtures submitted for the HPCCC separations.
Peak no. Compound Symbol Rt [min] max [nm] m/z [M+H]
+
m/z fromMS/MS of [M+H]
+
1 Betanin
a
Bt 14.3 538 551 389
2 17-Decarboxy-betanin
a
17-dBt 15.1 505 507 345
1

Isobetanin
a
IBt 15.6 538 551 389
2

17-Decarboxy-isobetanin
a
17-dIBt 16.5 505 507 345
3

2-Decarboxy-isobetanin
a
2-dIBt 18.7 533 507 345
3 2-Decarboxy-betanin
a
2-dBt 18.7 533 507 345
4 2,17-Bidecarboxy-betanin
a
2,17-dBt 20.2 507 463 301
4

2,17-Bidecarboxy-isobetanin
a
2,17-dIBt 20.2 507 463 301
5 17-Decarboxy-neobetanin
b
17-dNBt 20.5 446 505 343; 299; 255
6 2,15,17-Tridecarboxy-2,3-dehydro-neobetanin
b
2,15,17-dec-2,3-dHNBt 21.3 394 415 253
7 14,15-Dehydro-betanin (neobetanin)
a
NBt 22.0 468 549 387; 343
8 2,17-bidecarboxy-2,3-dehydro-neobetanin
b
2,17-dec-2,3-dHNBt 22.8 409 459 297
9 2,15,17-Tridecarboxy-neobetanin
b
2,15,17-dNBt 23.5 451 417 255
10 2-Decarboxy-neobetanin
b
2-dNBt 26.0 483 505 343; 299; 255
a
Pigments fromthe mixture 1.
b
Pigments fromthe mixture 2 (tentatively identied).
36m. The column was mounted in a cantilever rotor containing
a counterweight for balance when rotating. The distance between
the holder axis of the coil and the central axis of the instrument
was 50mm (revolution radius R). The maximum rotation speed
was 2049rpm (240g eld). The CCC machines were connected to
a thermostat, which enabled maintaining a constant temperature
during the separation process (20

C). During all CCC runs a K-501

Knauer (Berlin, Germany) pump, UV-ViS detector Shimadzu (Lyon,
France) andfractioncollector FoxyJr. fromKnauer company(Berlin,
Germany) were used.
The positive ion electrospray mass spectra were recorded on a
ThermoFinnigan LCQ Advantage (electrospray voltage 4.5kV; cap-
illary 250

C; sheath gas: N
2
) coupled to a ThermoFinnigan LC
Surveyor pump utilizing the HPLC systems. The MS was controlled
andtotal ionchromatograms andmass spectra wererecordedusing
ThermoFinnigan Xcalibur software (San Jose, CA, USA). Heliumwas
used to improve trapping efciency and as the collision gas for
CID experiments. The relative collision energies for MS/MS anal-
yses were set at 30% (according to a relative energy scale). For the
LCMS/MS analyses, a 25cm3.0mm, 5m Luna C18 (2) Pheno-
menex chromatographic column was used.
HPLC analyses were carried out using a Gynkotek HPLC system
with UVD340U Gynkotek HPLC Pump Series LPG-3400A and ther-
mostat (Gynkotek Separations, H. I. Ambacht, The Netherlands).
The software package Chromeleon 6.0 (Gynkotek Separations) was
applied for the data acquisition. For the CCC fraction analysis by
HPLC, a 10cm2.1mm, 2.7mSupelco (C-18) column was used.
2.4. Solvent systems
The solvent systems initially investigated for the HPCCC sepa-
ration are listed in Table 2 and were divided into two groups: A
highly polar solvent systems containing ammonium sulphate salt
(AIAIII) and B ion-pair solvent systems containing an ion-pair
agent (BIBIV). The ion-pair solvent systems were prepared in a
separator funnel by mixing appropriate solvents then, after equil-
ibration, the phases were separated and sonicated before HPCCC
separations.
The biphasic highly polar solvent systems, containing saturated
ammoniumsulphatesolution, werepreparedas describedbyFahey
et al. [19]. Saturated ammoniumsulphate was made by dissolving
the salt in boiling water, letting this cool down to 78

C and decant-
ing the supernatant. The saturated ammonium sulphate was then
mixed with the remaining solvents in the ratio described in Table 2.
The solvent systems were equilibrated at 20

C and then the phases

were separated and sonicated in order to remove dissolved gases.
2.5. Separation of betalains by HPCCC
Determination of stationary phase retention for each solvent
systemand preliminary separation studies were performed on the
analytical Mini HPCCC instrument. The solvent systems were pre-
paredaccording to Section2.4. The Mini HPCCCinstrument was run
at aowrateof 0.25ml/mininbothnormal phase(systems AIAIII)
and reversed phase (systems BIBIV) modes. The sample (15mg)
was dissolved in 1.5ml of stationary phase (systems AIAIII) or
mobile phase (systems BIBIV). The choice of the injection solvent
was primarily a result of betalain solubility. The chromatographic
column was rst entirely lled with the stationary phase and the
mobile phase was pumped while the coil was rotating at 2049rpm
at constant temperature of 20

C. The retention of the stationary

phase measured for each solvent systems was as follows: 64.3%
(systemAI), 42.3%(systemAII) and50.0%(systemAIII), 52.6%(sys-
temBI), 49.0% (systemBII), 69.8% (systemBIII), and 60.5% (system
BIV).
The semi-preparative separation of the betalain mixtures was
performed on the Spectrum HPCCC. Using either solvent system
AI or BIV, the centrifuge was run at a ow rate of 1.0ml/min.
Table 2
Composition of the solvent systems tested for betalains separation by HPCCC.
Systemno. Composition
A Highly polar solvent systems with salt
I 1-PrOHACNsaturated (NH
4
)
2
SO
4
H
2
O (v/v/v/v, 1:0.5:1.2:1
II EtOHACN-1PrOHsaturated (NH
4
)
2
SO
4
H
2
O (v/v/v/v/v, 0.5:0.5:0.5:1.2:1)
III EtOH-1BuOHACNsaturated (NH
4
)
2
SO
4
H
2
O (v/v/v/v/v, 0.5:0.5:0.5:1.2:1)
B Ion-pair solvent systems
I TBME-1BuOHACNH
2
O (0.7% TFA) (v/v/v/v, 2:2:1:5)
II TBME-1BuOHACNH
2
O (1.0% TFA) (v/v/v/v, 2:2:1:5)
III TBME-1BuOHACNH
2
O (0.4% HFBA) (v/v/v/v, 2:2:1:5)
IV TBME-1BuOHACNH
2
O (0.7% HFBA) (v/v/v/v, 2:2:1:5)
A. Sprna-Kucab et al. / J. Chromatogr. B 941 (2013) 5461 57
The sample (15mg) was dissolved in 1.5ml of stationary phase
(system AI) or mobile phase (system BIV). As with the Mini
CCC runs, the chromatographic coil was rst entirely lled with
the stationary phase and the mobile phase was pumped while
the coil was rotating at 1600rpm at constant temperature of
20

C. The retention of the stationary phase was measured on

the Spectrum instrument as follows: 80.5% (system AI) and
81.2% (system BIV). The system AI enabled purication of
2-decarboxy-betanin/-isobetanin (3.24mg), 2,17-bidecarboxy-
betanin/-isobetanin (3.42mg) and neobetanin (0.47mg) (mixture
1) plus 17-decarboxy-neobetanin (1.65mg), 2,15,17-tridecarboxy-
2,3-dehydro-neobetanin (1.88mg), 2-decarboxy-neobetanin
(6.39mg) and2,15,17-tridecarboxy-neobetanin(0.87mg) (mixture
2) and the system BIV was effective for 2,17-bidecarboxy-
betanin/-isobetanin (3.74mg) and neobetanin (0.44mg) (mixture
1) plus 2,15,17-tridecarboxy-2,3-dehydro-neobetanin (2.4mg),
2,17-bidecarboxy-2,3-dehydro-neobetanin (1.5mg) and 2,15,17-
tridecarboxy-neobetanin (1.2mg) (mixture 2).
The efuent fromthe outlet of the HPCCC was monitored using
a UV-ViS detector (Gilson, Middleton, WI, USA) and collected into
test tubes with a fraction collector at 6min intervals (ow rate
0.25ml/min and 1ml/min). The elution-mode was stopped when
all the pigments had been eluted as shown by the UV-ViS detector.
Where necessary, this was followed by the extrusion-mode with
the pumping of the stationary phase at a ow rate 0.5ml/min
(analytical scale) and 2.0ml/min (semi-prep scale) during the coil
rotation.
2.6. HPLC analysis (LC-DADESIMS/MS)
To prevent dissolved salt in the fractions fromadversely affect-
ing the HPLC analysis, a precipitation of the salt bulk from the
samples was accomplished with methanol. LC-DAD analyses of
mixtures 1 and 2 and HPCCC fractions were carried out using a
gradient elution mode at 40

C with methanol (A) and 2% aqueous

formic acid (B) system: 5% A in B at 0min, a gradient to 7% A in B at
2min and 20% A in B at 8min then 40% A in B at 10min and 80% A
in B at 12min, returning to the start conditions in 0.6min. For the
LCMS/MS analyses, a solvent system: 7% A in B at 0min a gradient
to 30% A in B at 35min (A, methanol; B, 2% formic acid in water)
was used. The injection volume was 70l and the ow rate was
0.5ml/min (LC-DAD and LCMS/MS systems).
2.7. Freeze drying
The HPCCC fractions were diluted with deionised water because
they contained large amounts of solvents. The diluted fractions
were then frozen and lyophilized. The fractions containing higher
amounts of solvents were partially evaporated by speed vacuum
centrifuge at room temperature to minimize compound degrada-
tion and then freeze dried.
3. Results and discussion
3.1. Analysis of decarboxylated and dehydrogenated derivatives
of betanin/isobetanin
For the experiments, two different mixtures of betanin and
its derivatives, differing in decarboxylation and dehydrogenation
products, were obtained as a result of the different heating times
of the acidied betanin extract (Table 1). Mixture 1 contained
mainly betanin, isobetanin, neobetanin and decarboxy-betanins
while mixture 2 consisted of decarboxy- and dehydro-betanins.
The mixtures differed in the pigment polarities and physico-
chemical properties determining their chromatographic behavior.
For example, the compounds in mixture 1 were more unsta-
ble than in mixture 2 and the HPLC retention times of the
decarboxylated and dehydrogenated derivatives were longer in
comparison to their corresponding betacyanins, due to their
lower polarity. Betanin, as well as 2-, 17-, and 2,17-bidecarboxy-
betanins detected by HPLC and LCMS/MS were identied
according to the standards isolated in previous studies [20],
and were monitored according to their retention times, and ViS
absorption maxima
max
(538, 533, 505, 507nm for betanin,
2-monodecarboxy-, 17-monodecarboxy- and 2,17-bidecarboxy-
betanins, respectively). The other compounds were mostly
tentatively identied based on their
max
(446, 394, 468, 409, 451,
483nm for 17-decarboxy-neobetanin, 2,15,17-tridecarboxy-2,3-
dehydro-neobetanin, neobetanin, 2,17-bidecarboxy-2,3-dehydro-
neobetanin, 2,15,17-tridecarboxy-neobetanin and 2-decarboxy-
neobetanin, respectively) as well as their protonatedmolecular and
fragmentation ions (Table 1) according to a previous discussion
[21,22].
3.2. HPCCC separations of betacyanins and their derivatives
This study is a rst attempt of a complete HPCCC separation
of betacyanins and their decarboxylated/dehydrogenated deriva-
tives obtained during thermal treatment of red beet juice. Finding
an appropriate phase system for the successful CCC separation of
polar betacyanins is problematic [69]. However, studies on beta-
lains from Phytolacca americana [6] and Bougainvillea glabra [9]
suggested that an effective separation of the less polar compounds
(e.g. acylated-betacyanins) could be achieved in solvent systems
with ion-pair reagents.
The use of hydrophilic solvent systems containing ammonium
sulphate for the purication of anionic glucosinolates from crude
plant homogenates [19] suggested that they were appropriate sys-
tems for a puricationof polar compounds, however a similar polar
system consisting of EtOHACN(NH
4
)
2
SO
4
(satd. soln)water
(1:0.5:1.2:1, v/v/v/v) was unsuccessfully used for separation of
betanin and isobetanin [11] as these compounds were co-eluted.
In order to investigate the separation of newbetacyanin groups
(the decarboxylated and dehydrogenated derivatives) by HPCCC,
both types of solvent systems were tested, i.e.:
(A) Highly polar solvent systems containing a high concentration
of ammoniumsulphate to enable the formation of two phases
in solvent systems containing water in both phases.
(B) Ion-pair, aqueous-organic solvent systems including ion-pair
reagents (TFA, HFBA).
3.2.1. Highly polar solvent systems containing ammonium
sulphate
3.2.1.1. Analytical scale separation of decarboxylated and dehydro-
genated betanins. The initial experiments were carried out in an
analytical machine with highly polar solvent systems containing
ammonium sulphate (Table 2, systems AIAIII). The pH of these
solvent systems is ca. 5.5 and at this pH betalains are more sta-
ble. The results of the separation of decarboxylated (Fig. 2) and
dehydrogenated (Fig. 3) betanins in the three solvent systems are
compared. In this mode of separation (tail-to-head), the mobile
phase is the upper phase (organic phase) and the stationary phase
is the lower phase (aqueous phase), therefore, the more hydropho-
bic compounds are eluted rst as expected. The high concentration
of ammoniumsulphate in the aqueous phase enhances the reten-
tion of the stationary phase in CCC by increasing the difference in
density between the two phases. The stationary phase retention in
CCC inuences peak resolution; the larger amount of the station-
ary phase in the coil the higher resolution. In systemAI, the highly
polar Bt/IBt (1/1

) are retained longer in the HPCCC coil and are

58 A. Sprna-Kucab et al. / J. Chromatogr. B 941 (2013) 5461
Fig. 2. Reconstructed HPLC chromatograms of betalains (mixture 1) in three high
salt-solvent systems separated by analytical HPCCC (composition of the solvent
systems, see Table 2).
eluted with 17-dBt/-dIBt (2/2

) during elution extrusion process

(Fig. 2). The separation of NBt (7) is very successful in the applied
conditions. 2,17-dBt/-dIBt (4/4

) are eluted as the rst decarboxy-

betacyanins, partially resolved from2-dBt/-dIBt (3/3

). In the group
of decarboxylated betacyanins (mixture 1) separated in solvent
system AII, 2,17-dBt/-dIBt (4/4

) are eluted as rst, virtually coin-

cident with 2-dBt/-dIBt (3/3

). Neobetanin (7) is eluted next and

is relatively pure. Yet the nal four compounds 17-dBt/-dIBt (2/2

)
and Bt/IBt (1/1

) showa considerable peak overlap, with none being

pure. SystemAIII gives good results for 2,17-dBt/-dIBt (4/4

) and 2-
dBt/-dIBt (3/3

) separation, however, 17-dBt/-dIBt (2/2

) and Bt/IBt
(1/1

) are eluted during elution extrusion process less separated

than in systemAI (Fig. 2). The elution order of betalains is the same
for all solvent systems and mainly depends on their polarity. The
more hydrophobic compounds are eluted rst, followed by more
polar pigments. The applied solvent systems have different polar-
ity, the most polar being system AII, then system AI and system
AIII. In systemAII, betalains are eluted too fast and therefore with
poor resolution. Neobetanin (7) is eluted signicantly earlier in the
most polar systemAII than in the remaining systems. In the case of
2,17-dBt/-dIBt (4/4

) and 2-dBt/-dIBt (3/3

), the situation is similar.

The separation of these pigments clearly depends on the polarity of
the solvent systems and is more effective in less polar solvent sys-
tems AI andAIII. The efciency of separationis also associatedwith
retention of the stationary phase, which is the highest in systems
Fig. 3. Reconstructed HPLC chromatograms of betalains (mixture 2) in three high
salt-solvent systems separated by analytical HPCCC (composition of the solvent
systems, see Table 2).
AI and AIII. The retention of the stationary phase and polarity of
the solvents presumably inuence separation of 17-dBt/-dIBt (2/2

)
fromBt/IBt (1/1

) which is the most effective in solvent systemAI.

In this case, the retention of the stationary phase is the highest
(64.3%).
The separation of dehydrogenated betacyanins (mixture 2) by
analytical HPCCCis presentedinFig. 3. The best results are obtained
for systems AI and AIII where the separation of the majority of
dehydro-derivatives is quite effective, with a very good separation
of 17-dNBt (5) from the rest of the compounds, as a result of the
highest stationaryphaseretentioninthesesolvent systems anddue
to lower polarity of 5. In solvent systemAI, only a partial overlap
in the group of 2,15,17-dec-2,3-dHNBt (6), 2,17-dec-2,3-dHNBt (8),
2,15,17-dNBt (9), and 2-dNBt (10) is observed. Solvent systemAII,
except for 17-dNBt (5), gives no pure fractions, with the other four
peaks substantially overlapped. In this group of compounds tested
in system AIII, only a relatively good separation of 2,15,17-dec-
2,3-dHNBt (6) and 2,15,17-dNBt (9) is observed. In systemAII, the
pigments are mostly co-eluted except of 17-dNBt (5) in spite of
its early elution with the other compounds. The best results are
obtained for systemI where the retention of the stationary is the
highest (Fig. 3).
3.2.1.2. Semi-preparative scale separation of decarboxylated and
dehydrogenated betanins. The systemAI was used to separate mix-
tures 1and2usingasemi-preparativemachine. Theappliedsolvent
A. Sprna-Kucab et al. / J. Chromatogr. B 941 (2013) 5461 59
Fig. 4. HPCCCchromatogramof betalains (mixture1) after theseparationinthehigh
salt-solvent system by semi-prep HPCCC (system AI, see Table 2). Peak numbers
refer to compounds shown in Table 1.
system enables a signicantly better separation of betalains due
to a higher retention of the stationary phase (80.5%). The HPCCC
instrument used has almost twice the coil length of the analyti-
cal instrument, plus a wide bore (1.6mm) to reduce any plug ow
effects. The HPCCCchromatogrammonitoredat
500nm
(systemAI)
for mixture 1 is shown in Fig. 4. The rst two peaks (2,17-dBt/-dIBt
(4/4

) and 2-dBt/-dIBt (3/3

)) are partially resolved with a resolu-

tion of 0.67 and the third compound (NBt (7)) is eluted as a single
peak. Themost polar compoundpairs (17-dBt/-dIBt (2/2

) andBt/IBt
(1/1

)) appear to co-elute in fractions 4 and 5 (elution extrusion

mode). However, the reconstructed HPCCC chromatogram(Fig. 5A)
of mixture 1 shows a tendency of a separation of the pairs 1/1

and
2/2

. The extrusion of the column content results in fractions highly

rich in either Bt/IBt (1/1

) or 17-dBt/-dIBt (2/2

) (Fig. 5A). Compar-

ison of Fig. 2 and Fig. 5A and B demonstrates that polarity of the
solvent systems is the main factor determining the resolution of
the compounds. The separation of mixture 1 is not much effective
despite considerably higher retention of the stationary phase on
SpectrumCCC. The HPLCchromatograms of the crude mixtures and
the puried fractions are depicted in Fig. 6.
Fig. 7 demonstrates the HPCCC chromatogram of mixture 2
(monitored at
500nm
) separated in system AI with only partial
Fig. 6. HPLC chromatograms of betalains (mixture 1) before separation by semi-
prep HPCCC (a) and selected fractions after the separation in the high salt-solvent
system(AI, see Table 2) (bf).
Fig. 7. HPCCCchromatogramof betalains (mixture2) after theseparationinthehigh
salt-solvent system by semi-prep HPCCC (system AI, see Table 2). Peak numbers
refer to compounds shown in Table 1.
Fig. 5. Reconstructed HPLC chromatograms of betalains after the separation in the high salt-solvent system(a mixture 1, solvent systemAI, b mixture 2, solvent system
AI) and ion-pair solvent systemwith 0.7% HFBA (c mixture 1, solvent systemBIV, d mixture 2, solvent systemBIV, see Table 2) by semi-prep HPCCC.
60 A. Sprna-Kucab et al. / J. Chromatogr. B 941 (2013) 5461
Fig. 8. HPLC chromatogramof betalains (mixture 2) before separation by semi-prep
HPCCC (a) and selected fractions after the separation in the high salt-solvent system
(AI, see Table 2) (bf).
overlap of the rst four components and a complete separa-
tion of 17-dNBt (5) eluted as a single irregular broad peak. Two
partially resolved dehydrogenated betacyanins (2-dNBt (10) and
2,17-dec-2,3-dHNBt (8)) are eluted in the rst and second peak,
respectively, and are well separated from 2,15,17-dec-2,3-dHNBt
(6) and 2,15,17-dNBt (9). Fig. 8 shows the HPLC chromatogram of
the crude mixture 2 plus selected fractions fromthe HPCCC puri-
cation run.
3.2.2. HPCCC solvent systems containing ion-pair reagents (TFA,
HFBA)
In this study, four solvent systems (Table 2, systems BIBIV)
containing TFA or HFBA were compared. The presence of TFA or
HFBA in the solvent systems at different concentrations inuences
the stationary phase retention (which is higher at a lower concen-
tration of the acids).
The best separation results for mixture 1 (Fig. 5C) were obtained
in the systemwith 0.7% HFBA (systemBIV) (despite a smaller sta-
tionary phase retention than in the systemwith 0.4%HFBA(system
BIII)). Comparison of systems BIII and BIV (data not shown) leads
to a conclusion that the amount of acid is a more signicant factor
determining the resolution of the compounds than the retention
of the stationary phase. Most of betalains from mixture 1 are not
separated with solvent systems BI, BII nor BIII at all (data not
shown). TFA forms less lipophilic ion-pairs than HFBA with beta-
lains which are eluted too early from the coil which makes their
effective separation impossible. For further experiments on semi-
preparative scale, only system BIV was taken. The separation of
the dehydrogenated pigments (mixture 2) on analytical scale is not
successful in the solvent systems containing TFA (systems BI-BII)
and all compounds are co-eluted (data not shown). HFBA creates
more lipophilic ion-pairs than TFA, therefore, the presence of HFBA
signicantly shifts the analytes to the organic phase, improving
K
D
values. However, comparing the systems BIII and BIV reveals
that only systemBIV could be useful for the dehydrogenated beta-
cyanins separation (data not shown), considering that the amount
of acid inuences betalains separation.
Based on the initial results obtained for the analytical systems,
systemBIVwas usedona semi-preparative scale for the separation
of the two groups of betacyanins (the retention of the stationary
phase is 81.2%inthe operated apparatus). For the mixture 1, almost
pure fractions are obtained for the less polar neobetanin (7) and for
Fig. 9. HPLC chromatogramof betalains (mixture 1) before separation by semi-prep
HPCCC (a) and selected fractions after the separation in the ion-pair solvent system
with 0.7% HFBA (BIV, see Table 2) (be).
the more polar 2,17-dBt/-dIBt (4/4

) (Fig. 5C). It can be noticed that

neobetanin (7) is eluted much faster than betanin/isobetanin (1/1

)
and decarboxy-betacyanins (2/2

, 3/3

, 4/4

). The faster elution of

neobetanin (7) results fromthe weaker formation of ion-pairs with
the anions due to a lower protonation of its structure.
Bt/IBt (1/1

) is eluted with just a minor contamination fromNBt

(7), however, its tailing peak co-elutes with unresolved pairs of 17-
dBt/-dIBt (2/2

) and 2-dBt/-dIBt (3/3

) (Fig. 5C). For the purication

of 17-dBt/-dIBt (2/2

) and 2-dBt/-dIBt (3/3

) the high salt system

AI (Fig. 5A) is recommended instead. The HPLC chromatograms of
the crude injection material and selected puried fractions from
mixture 1 can be seen in Fig. 9.
Comparing Figs. 5C and 9, the elution proles of betalains
obtained from the CCC with ion-pair solvent systems (reversed
mode) are completely different from the proles observed in the
HPLC system (working also in the reversed mode). For mixture 1,
the following elution order in the HPLC system (Fig. 9) is usually
observed: Bt (1), 17-dBt (2), IBt (1

), 17-dIBt (2

), 2-dBt/-dIBt (3/3

),
2,17-dBt/-dIBt (4/4

) and NBt (7), whereas in the CCC system it is:

7, 1/1

, 2/2

, 3/3

, and 4/4

(Fig. 5C), indicating that 1/1

and 2/2

are
eluted as pairs in contrast to HPLC elution. The studied differences
result from different effectiveness of the interactions between
selected betalains and the ion-pair reagents, which inuences their
separation and elution order. In particular, the differences in ion-
ization properties are observed between the decarboxylated and
dehydrogenated betacyanins (e.g. very fast elution of NBt (7)). Elu-
tion of betalains in HPLC is based on their polarity, the more polar
pigments are eluted as rst. In CCC the situation is similar, more
polar betanins and decarboxy-betanins are eluted depending on
their polarity except NBt (7). Neobetanin (less positively charged
pigment) (7) presumably does not create stabile ion-pairs with
HFBA, therefore, its polarity is not signicantly changed during the
CCC separation.
For mixture 2, a goodseparationof dehydrogenatedbetacyanins
is observed in systemBIV (Fig. 5D) on the semi-preparative scale.
Interestingly, the compounds are eluted in different order than in
the high salt system AI (Fig. 5B). The different elution order is
observed due to different separation modes (tail-to-head versus
head-to-tail) but also because of a formation of ion-pairs with
betalains. Especially a completely different relative retention is
observed for 17-dNBt (5) and 2,15,17-dec-2,3-dHNBt (6) in both
the systems. Only a slightly higher overlap is observed for 17-dNBt
(5) and2-dNBt (10) insystemBIVanda goodseparationof 2,15,17-
dec-2,3-dHNBt (6), 2,17-dec-2,3-dHNBt (8) and 2,15,17-dNBt (9) is
accomplished.
A. Sprna-Kucab et al. / J. Chromatogr. B 941 (2013) 5461 61
Fig. 10. HPLC chromatogram of betalains (mixture 2) before separation by semi-
prep HPCCC(a) andselectedfractions after the separationinion-pair solvent system
with 0.7% HFBA (BIV, see Table 2) (bf).
Fig. 10 shows the HPLC chromatograms of the crude mixture 2
together with selected fractions of the pure components. As in the
case of mixture 1, the elution order of dehydrogenated betacyanins
in the CCC system (Fig. 5D) is different from their elution order in
the RP-HPLC system (Fig. 10). In the CCC system, early elution of
2-dNBt (10) is observed, which in principle is eluted very late in
the RP-HPLC systems. In addition, the elution of 2,15,17-dec-2,3-
dHNBt (6) is very late in the CCC system, contrasting with a fast
elution in RP-HPLC.
4. Conclusions
In this study, we have shown that the separation of betalains
using highly polar solvent systems is possible and very effective for
selected structures of betalains: 2-decarboxy-betanin/-isobetanin,
2,17-bidecarboxy-betanin/-isobetaninandneobetanin(mixture 1),
and17-decarboxy-neobetanin, 2,15,17-tridecarboxy-2,3-dehydro-
neobetanin, 2,17-bidecarboxy-2,3-dehydro-neobetanin, 2,15,17-
tridecarboxy-neobetanin and 2-decarboxy-neobetanin (mixture
2). This is the rst report on preparative isolation of the mentioned
compounds, using HPCCC with highly polar solvent systems.
The study conrms that ion-pair solvent systems with HFBA are
much more effective than those with TFA because betalains cre-
ate more hydrophobic structures which are shifted to the organic
phase improving their K
D
values. Moreover, the acid concentration
inuences the stationary phase retention. Increasing concentration
of the acid can decrease differences of the density of the upper and
lower phases as well as can enhance creation of emulsions. In spite
of the lower stationary phase retention, the systemwith 0.7% HFBA
is more effective than the system with 0.4% HFBA for the separa-
tion of 2,17-bidecarboxy-betanin/-isobetanin, betanin/-isobetanin
and neobetanin (mixture 1) and 17-decarboxy-neobetanin,
2,15,17-tridecarboxy-2,3-dehydro-neobetanin, 2,17-bidecarboxy-
2,3-dehydro-neobetanin, 2-decarboxy-neobetanin and 2,15,17-
tridecarboxy-neobetanin (mixture 2).
In conclusion, the two solvent systems presented (highly polar
solvent systems containing ammonium sulphate salt and ion-pair
solvent systems) are capable of producing a number of pure com-
ponents with HPCCC of betalains, opening up the possibility of
utilizing these compounds commercially. Moreover, a combination
of the two CCC solvent systems together with RP-HPLC, results in
completelydifferent elutionorders andmakes themaveryversatile
tool for the isolation of a pigment on demand. The elution proles
for the CCC and HPLC runs are signicantly different, indicating
their different modes of separation.
In addition, the application of highly polar solvent systems
with ammonium sulphate salt containing no toxic peruorinated
acids is a rst step in the search for food-grade solvent systems,
which would be applied in the food, cosmetic and pharmaceutical
industries, taking advantage of betalains colorant, antioxidant and
possible chemopreventive properties.
Acknowledgements
The research was supported by the European Union through
the European Social Fund within CracowUniversity of Technology
development program top quality teaching for the prospective
Polish engineers; University of the 21st century project (contract
no.UDA-POKL.04.01.01-00-029/10-00).
The nancial support by the UKHigher Education Infrastructure
Fund (HEIF4) is also gratefully acknowledged.
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