Supplementary Data for
Isolation of (five) Steviol Glycosides from a Stevia Rebaudiana
Formulation by Gradient Elution Countercurrent
Chromatography
Michael Englert1*, Carolin Kaiser2, Wolfgang Schwack2, Walter Vetter1
1
University of Hohenheim, Institute of Food Chemistry, Department of Food Chemistry,
Garbenstrasse 28, D-70599 Stuttgart, Germany
2
University of Hohenheim, Institute of Food Chemistry, Department of Food Chemistry and
Analytical Chemistry, Garbenstrasse 28, D-70599 Stuttgart, Germany
* Corresponding author:
Michael Englert
Phone: +49 711 459 23527
Fax: +49 711 459 24377
e-mail: michael.englert@uni-hohenheim.de
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The supplementary data contains:
Fig. S1. Ternary phase diagram of the ethyl acetate/n-butanol/water solvent system.
Fig. S2. CCC-ELSD chromatogram of isocratic separation of stevia formulation with ethyl
acetate/n-butanol/water (3:2:5, v/v/v).
Fig. S3. CCC-ELSD chromatograms of the separation of stevia formulation using the ethyl
acetate/n-butanol/water solvent system with four different gradient programs.
Fig. S4. CCC-ELSD chromatogram n-hexane/n-butanol/water (1.5:3.5:5, v/v/v) of a mixture
of St and Reb C in isocratic mode with the solvent system according to X.-Y. Huang et al. [2].
Table S1. Most abundant ions observed in the total ion chronograms of steviol glycosides
during HPTLC/MS and corresponding monoisotopic masses.
Table S2. Reason for selection of solvent systems tested for isocratic elution CCC with
statement on the unsuitability and reason for rejection.
Table S3. Partitioning coefficients K of steviol glycosides in different compositions of the
ethyl acetate/n-butanol/water gradient system.
2
Fig. S1. Ternary phase diagram of the ethyl acetate/n-butanol/water solvent system [1] with
stationary phase (SP) and six different compositions A-F of the mobile phase. The tie-lines
indicated that the system can be used for gradient elution CCC as they originated in the SP.
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Figure S2. CCC-ELSD chromatogram obtained during the isocratic separation of 40 mg
(c = 10 mg/mL) stevia formulation with ethyl acetate/n-butanol/water (3:2:5, v/v/v) in tail-tohead mode with 1 mL/min with a Sf value of 70%.
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Figure S3. CCC-ELSD chromatograms (left side) obtained during the separation of about 40
mg (c = 10 mg/mL) stevia formulation extract using the ethyl acetate/n-butanol/water solvent
system and a mobile flow rate of 1 mL/min with four different gradient programs a)-d) (right
side).
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Figure S4. CCC-ELSD chromatogram obtained during the separation of 10.3 mg of a mixture
of St and Reb C in isocratic mode with the solvent system n-hexane/n-butanol/water
(1.5:3.5:5, v/v/v) according to X.-Y. Huang et al. [3].
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Table S1. Most abundant ions observed in the total ion chromatograms of steviol glycosides
during HPTLC/MS according to Morlock et. al [2] and corresponding monoisotopic masses.
Steviol glycoside
Monoisotopic mass
m/z
m/z
[M+Na]+
[M-H]+
StB
642.3251
665
641
Reb B
804.3780
827
803
St
804.3780
827
803
Reb C
958.4359
981
957
Reb A
966.4308
989
965
Dulc A
788.3831
811
787
Reb D
1128.4836
1151
1127
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Table S2. Reason for selection of solvent systems tested for isocratic elution CCC with
statement on the unsuitability and reason for rejection.
Solvent system Composition
Reason for selection
System A
n-hexane/n-BuOH/
Described
H2O (1.5:3.5:5)
literature [4] for the coefficients for Dulc A
in
Reason for rejection
the Unsuitable
partitioning
separation
of
three (K<0.1) and for StB (K>10)
steviol
glycosides and immediate flooding of
Reb A, Reb C and St
the stationary phase after
injection
System B
TBME/ACN/
Described
H2O (25:40:25)
literature
in
[5]
substances
intermediate
the Low
partitioning
for coefficients
for
all
of compounds
(K<0.5)
and
polarity insufficient retention of the
and promised a good stationary
phase
before
solubility of the sample sample injection and a total
in both phases
loss of stationary phase
after sample injection
System C
EtOAc/n-PrOH/
Was selected because of Low partitioning
H2O (7:3:1)
the absence of n-butanol coefficients for compounds
which was assumed to (K<0.5) except for StB and
be responsible for the almost no retention of
System D
EtOAc/n-BuOH/
instability of System A
stationary phase could be
[4]
observed
A suitable range of Low selectivity factor α
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H2O (3:2:5)
partitioning coefficients between Dulc A and Reb B
except for StB, a good and St and Reb C
solubility in this solvent
system and the initial
stationary
retention
phase
was
acceptable
Abbreviations: n-BuOH: n-butanol; n-PrOH: n-propanol; EtOAC: ethyl acetate
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Table S3. Partitioning coefficients K of steviol glycosides in different compositions A-F of
the ethyl acetate/n-butanol/water gradient system.
A
B
C
D
E
F
EtOAC/H2O/n-BuOH
77:8:15
62:10:28
54:11:35
48:12:40
40:14:46
13:17:70
StB
0.78
1.42
1.99
2.56
3.43
9.61
Reb A
0.15
0.40
0.57
0.82
1.21
5.30
Dulc A
0.63
0.99
1.56
2.10
2.87
8.50
St
0.41
0.89
1.34
1.85
2.32
7.49
Reb C
0.28
0.70
0.99
1.44
1.83
6.72
Reb B
0.20
0.57
0.79
0.96
1.44
6.00
Reb D
0.05
0.25
0.44
0.60
0.88
3.70
Abbreviations: n-BuOH: n-butanol; EtOAC: ethyl acetate
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References
[1] A.P. Foucault, Centrifugal Partition Chromatography, Chromatographic Science Series,
Vol 68. Marcel Dekker, New York, 1995.
[2] G.E. Morlock, S. Meyer, B.F. Zimmermann, J. Roussel, High-performance thin-layer
chromatography analysis of steviol glycosides in Stevia formulations and sugar-free food
products, and benchmarking with (ultra) high-performance liquid chromatography, J.
Chromatogr. A 1350 (2014) 102-111.
[3] X. Huang, J. Fu, D. Di, Preparative isolation and purification of steviol glycosides from
Stevia rebaudiana Bertoni using high-speed counter-current chromatography, Sep. Purif.
Technol. 71 (2010) 220-224.
[4] S.J. Gluck, M.P. Wingeier, Development of a phase system for intermediate polarity
compounds in centrifugal partition chromatography, J. Chromatogr. A 547 (1991) 69-78.
[5] A. Berthod, Practical approach to high-speed counter-current chromatography, J.
Chromatogr. A 550 (1991) 677-693.
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