Separation of Delphinidin-3-O-sambubioside, Cyanidin-3-O-sambubioside and p-Coumaric Acid from Cranberry by CCC Followed by prep-HPLC Using Robotic CCC Solvent System Selection

High-performance counter-current chromatography has been used for the separation of delphinidin-3-O-sambubioside, cyanidin-3-O-sambubioside and p-coumaric acid from crude extract of cranberry. The separation was performed with a two-phase solvent system composed of butanol/0.05% aqueous trifluoroacetic acid/methanol at a volume ratio of 4:5:1. The two-phase solvent system was selected following the determination of partition coefficients (K) in a range of solvent systems using a robotic solvent system selection method. Analytical scale CCC confirmed that this phase system separated the components from a crude cranberry extract (40 mg scale) with acceptable purities. Preparative CCC of 400 mg of crude yielded 4.2 mg of p-coumaric acid at a purity of over 98%, 3.6 mg of delphinidin-3-O-sambubioside at a purity of over 97% and 4.5 mg of cyanidin-3-O-sambubioside at a purity of 73%, which was further purified by preparative high-performance liquid chromatography to yield 3 mg cyanidin-3-O-sambubioside at 95% purity. The identification of delphinidin-3-O-sambubioside, cyanidin-3-O-sambubioside and p-coumaric acid was performed by ESI-MS, 1H-NMR and 13C-NMR spectra.