Separation of acylated anthocyanin pigments by high speed CCC

The reddish pigments of red onions (Allium cepa) and flowers of the tulip c ultivar Los Angelos, including fragile anthocyanins with aliphatic acylatio n, were separated in semi-preparative scale by high speed countercurrent ch romatography, HSCCC. The lower, polar phase of the biphasic solvent, t-buty l-methylether:n-butanol:acetonitrile:water (2:2:1:5 v/v) acidified with 0.0 1% trifluoroacetic acid, was used as mobile phase. Ail the five major antho cyanins in red onion, which contained the same aglycone (cyanidin), were se parated from a 750 mg anthocyanin-enriched isolate. Relative to cyanidin 3- glucoside, analogous anthocyanins with additional acyl group(s) (malonyl) a nd 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"-rhamnosylglucosid es) of delphinidin, cyaniding, and pelargonidin, and the 3-O-[6"-(2"'-acety lrhamnosyl)glucosides] of cyanidin and pelargonidin. Acetylated anthocyanin s were eluted after their non-acetylated analogues. Within each of the two groups, acetylated and non-acetylated, individual anthocyanins were separat ed according to their number of hydroxy-groups on the aglycone B-rings; hig hest number entailed lowest elution volume. All the acylated anthocyanins i n the anthocyanin-enriched isolates of both red onions and tulip "Los Angel os" were nearly baseline separated.