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.