SEPARATION OF ACYLATED ANTHOCYANIN PIGMENTS BY HIGH SPEED CCC

Abstract

The reddish pigments of red onions (Allium cepa) and flowers of the tulip cultivar Los Angelos, including fragile anthocyanins with aliphatic acylation, were separated in semi-preparative scale by high speed countercurrent chromatography, HSCCC. The lower, polar phase of the biphasic solvent, t-butyl-methylether:n-butanol: acetonitrile:water (2:2:1:5 v/v) acidified with 0.01% trifluoroacetic acid, was used as mobile phase. All the five major anthocyanins in red onion, which contained the same aglycone (cyanidin), were separated from a 750 mg anthocyanin-enriched isolate. Relative to cyanidin 3-glucoside, analogous anthocyanins with additional acyl group(s) (malonyl) and sugar (glucosyl) entailed higher and lower elution volume, respectively. The major pigments in a 430 mg anthocyanin-enriched sample from flowers of the tulip “Los Angelos” were determined to be the 3-O-(6″-rhamnosylglucosides) of delphinidin, cyaniding, and pelargonidin, and the 3-O-[6″-(2‴-acetylrhamnosyl)glucosides] of cyanidin and pelargonidin. Acetylated anthocyanins were eluted after their non-acetylated analogues. Within each of the two groups, acetylated and non-acetylated, individual anthocyanins were separated according to their number of hydroxy-groups on the aglycone B-rings; highest number entailed lowest elution volume. All the acylated anthocyanins in the anthocyaninenriched isolates of both red onions and tulip “Los Angelos” were nearly baseline separated.