Separation of amaranthine-type betacyanins by ion-pair high-speed countercurrent chromatography

• Betacyanins from Iresine lindenii leaves were separated by HSCCC for the first time.
• An orthogonality between the HSCCC and HPLC separation of betacyanins is observed.
• Betacyanins of a wide range of polarities were detected in the I. lindenii leaves extract.
• The separation of polar betacyanins was enabled at the presence of HFBA ion-pair reagent.
• New acylated betacyanins were identified.

Betacyanins, red-violet plant pigments, were fractionated by ion-pair high-speed countercurrent chromatography (IP-HSCCC) from leaves extract of Iresine lindenii Van Houtte, an ornamental plant of the family Amaranthaceae. An HSCCC solvent system consisting of TBME–1-BuOH–ACN–H2O (1:3:1:5, v/v/v/v) was applied using ion-pair forming heptafluorobutyric acid (HFBA). Significantly different elution profiles of betacyanin diastereomeric pairs (derivatives based on betanidin and isobetanidin) observed in the HSCCC in comparison to HPLC systems indicate a complementarity of both techniques’ fractionation capabilities. The numerous diastereomeric pairs can be selectively separated from each other using the HSCCC system simplifying the pigment purification process. Apart from the three well known highly abundant pigments (amaranthine, betanin and iresinin I) together with their isoforms, three new acylated (feruloylated and sinapoylated) betacyanins as well as known pigment hylocerenin (previously isolated from cacti fruits) were characterized in the plant for the first time and they are new for the whole Amaranthaceae family.