High-speed counter-current chromatography in separation of betacyanins from flowers of red Gomphrena globosa L. cultivars

•Betacyanins from flowers of Gomphrena globosa L. were separated for the first time by CCC.•No artefacts (additional decarboxylated derivatives of separated betacyanins) were detected.•The new, highly polar system was applied for the first time in CCC.•Effective separation of betanin in the new solvent system is reported.•Modification of the new system enables separation of betanin and acylated betacyanins.

Antioxidant and possible chemopreventive properties of betacyanins, natural plant pigments, contribute to a growing interest in their chemistry and separation. Mixtures of betacyanins from fresh red Gomphrena globosa L. cultivar flowers were separated in three highly polar solvent systems by high-speed counter-current chromatography (HSCCC) for a direct comparison of their separation effectiveness. Three samples of crude extract (600 mg) were run on semi-preparative scale in solvent system (NH4)2SO4soln - EtOH (2.0:1.0, v/v) (system I) and the modified systems: EtOH - ACN - 1-PrOH - (NH4)2SO4satd.soln - H2O (0.5:0.5:0.5:1.2:1.0, v/v/v/v/v) (system II) and EtOH - ACN - (NH4)2SO4satd.soln - H2O (1.0:0.5:1.2:1.0, v/v/v/v) (system III). The systems were used in the head-to-tail (system I) or tail-to-head (systems II and III) mode. The flow rate of the mobile phase was 2.0 ml/min and the column rotation speed was 860 rpm. The retention of the stationary phase was 52.0% (system I), 80.2% (systems II) and 82.0% (system III). The betacyanins in the crude extract as well as HSCCC fractions were analyzed by LC-MS/MS.System I was applied for the first time in HSCCC for the separation of betacyanins and was quite effective in separation of amaranthine and 17-decarboxy-amaranthine (αI = 1.19) and very effective for 17-decarboxy-amaranthine and betanin (αI = 2.20). Modification of system I with acetonitrile (system III) as well as acetonitrile and propanol (system II) increased their separation effectiveness. Systems II-III enable complete separation of 17-decarboxy-amaranthine (KD(II) = 2.94, KD(III) = 2.42) and betanin (KD(II) = 2.46, KD(III) = 1.10) as well as betanin and gomphrenin I (KD(II) = 1.62, KD(III) = 0.74). In addition, separation of amaranthine and 17-decarboxy-amaranthine is the most effective in system II, therefore, this system proved to be the most suitable for the separation of all polar betacyanins.