New solvent systems for gradient counter-current chromatography in separation of betanin and its derivatives from processed Beta vulgaris L. juice.

• Betalains were separated by CCC in new food-grade and gradient systems for the first time.
• The partition coefficients of betalains depend mostly on acid and ethanol gradients.
• The stationary phase retention for applied systems is very high (up to 80%).
• The lower ethanol or acid gradient the higher retention of the stationary phase.
• The low ethanol and acid gradient systems are more effective for betalains separation.

Betalains, natural plant pigments, are beneficial compounds due to their antioxidant and possible chemoprotective properties. A mixture of betalains: betanin/isobetanin, decarboxybetanins and neobetanin from processed red beet roots (Beta vulgaris L.) juice was separated in food-grade, gradient solvent systems using high-performance counter-current chromatography (HPCCC). The decarboxylated and dehydrogenated betanins were obtained by thermal degradation of betanin/isobetanin from processed B. vulgaris L. juice under mild conditions. Two solvent systems (differing in their composition by phosphoric acid and ethanol volume gradient) consisting of BuOH–EtOH–NaClsolution–H2O–H3PO4 (v/v/v/v/v, 1300:200–1000:1300:700:2.5–10) in the ‘tail-to-head’ mode were run. The flow rate of the mobile phase (organic phase) was 1.0 or 2.0 ml/min and the column rotation speed was 1600 rpm (20 °C). The retention of the solvent system stationary phase (aqueous phase) was ca. 80%. The system with the acid and ethanol volume gradient consisting of BuOH–EtOH–NaClsolution–H2O–H3PO4 (v/v/v/v/v, 1300:200–240:1300:700:2.5–4.5) pumped at 2.0 ml/min was the most effective for a separation of betanin/isobetanin, 17-decarboxy-betanin/-isobetanin, 2-decarboxy-betanin/-isobetanin, 2,17-bidecarboxy-betanin/-isobetanin pairs as well as neobetanin. The pigments were detected by LC–DAD and LC–MS. The results are crucial in the application of completely food-grade solvent systems in separation of food-grade compounds as well, and the systems can possibly be extended to other ionizable and polar compounds with potential health benefits. In particular, the method is applicable for the isolation and purification of betalains present in such rich sources as B. vulgaris L. roots as well as cacti fruits and Amaranthaceae flowering plants due to modification possibilities of the solvent systems polarity.