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 1 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. 3 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%. 4 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). 5 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]. 6 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 7 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 8 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 9 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 10 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. 11