High salt-solvent systems in separation of betanin and its derivatives from red beet (Beta vulgaris L.) by high-performance countercurrent chromatography (HPCCC)

A study on a separation of betanin and its decarboxy- and dehydro-derivatives obtained from red beet roots (Beta vulgaris L.) using analytical high-performance countercurrent chromatography (HPCCC — Dynamic Extractions Ltd., UK) was performed. The HPCCC process was accomplished in the ‘tail to head’ mode with three highly polar solvent systems with high salt concentrations: 1-propanol-acetonitrile-saturated ammonium sulphate-water (v/v/v/v, 1:0.5:1.2:1); ethanol-acetonitrile-1- propanol-saturated ammonium sulphate-water (v/v/v/v/v, 0.5:0.5:0.5:1.2:1) and ethanol-1-butanol-acetonitrile-saturated ammonium sulphate-water (volume ratio), 0.5:0.5:0.5:1.2:1). HPLC analysis was performed in a conventional reversed phase mode with diode-array (DAD) detection to characterize the composition of obtained fractions. The applied solvent systems enabled the separation of the betalain pigments with high efficiency for the first time. In the mode of separation selected, the more hydrophobic compounds eluted first as expected. Moreover, for the first time, the applied HPCCC solvent systems generated a separation of 2-decarboxy-betanin from 17- and 2,17-bidecarboxy-betanin as well as from neobetanin and betanin

Separation of betalains by gradient high-speed counter-current chromatography

A study on separation of betalain mixture obtained from red beet juice (Beta vulgaris L.) by analytical high-speed counter-current chromatography (HSCCC) was performed. The extract was obtained by thermal treatment of acidified red beet juice for 30 min in 85 °C. The pigment mixture consisted of betanin/isobetanin as well as their decarboxy- and dehydro-derivatives. The HSCCC process was accomplished in the ‘tail to head’ mode with two polar solvent systems containing salt: BuOH-EtOH-NaClsolution-H2OH3PO4 (1300:700-1000:1300:700:2.5-5.5 (system I), 1300:200-400:1300:700:2.5-4.5 (system II); v/v/v/v/v). The retention of the stationary phase was 73% (system I) and 79% (system II). The mobile phase was pumped at 2 ml/min flow rate. HPLC-DAD-ESI-MS analyses were performed in reversed phase mode for the obtained HSCCC fractions and crude extract. The solvent systems enabled separation of betanin and decarboxy-betanins (system I and II) as well as neobetanin (system II). Additionally, some pure fractions of 17-decarboxy-betanin and 2,17-bidecarboxy-betanin were obtained in system II

Research on solvent systems with tetra-n-butylammonium bromide for counter-current chromatography of betalains

In this study, new two-phase solvent systems for counter-current chromatography (CCC) consisting of n-butanol and water as well as various amounts of acetic acid, acetonitrile, ethanol, acetone or ethyl acetate were tested. Additionally, tetra-n-butylammonium bromide (TBAB) was introduced into the system in the form of aqueous solutions or phosphate-citrate buffer (pH 6.7) in order to form ion-pairs with betalains. The selection of buffer pH was based on their ability to create ion pairs by tetraalkylammonium salts, with selected betalains under these conditions. In this study, it is shown that the settling time of two phases is longer with the increase of acetic acid/acetonitrile/ethanol/acetone/ethyl acetate. For selected solvent systems with high amounts of acetonitrile, ethanol and acetone two phases were not observed. The systems with acetone have the largest increase of settling time. Ethyl acetate systems were characterized by a slow settling time increase. In systems containing additionally 2% aqueous TBAB, smaller changes in settling time than in similar systems without TBAB were observed. Addition of TBAB in the buffer resulted in a prolongation of settling time. Solvent systems in which the separation between the aqueous and organic phases was visually best, were selected from among all the tested systems and the betalain partition coefficients were measured by LC-DAD-ESI-MS. The best results were observed for systems: n-butanol-water-acetic acid (2:2.5:0.75, v/v/v), n-butanol-water-acetic acid (2:2.5:1, v/v/v) and n-butanol-TBAB in water-acetonitrile (2:2.5:0.5, v/v/v)