Simple solution for a complex problem: Proanthocyanidins, galloyl glucoses and ellagitannins fit on a single calibration curve in high performance-gel permeation chromatography

This study was undertaken to explore gel permeation chromatography (GPC) for estimating molecular weights of proanthocyanidin fractions isolated from sainfoin (Onobrychis viciifolia). The results were compared with data obtained by thiolytic degradation of the same fractions. Polystyrene, polyethylene glycol and polymethyl methacrylate standards were not suitable for estimating the molecular weights of underivatized proanthocyanidins. Therefore, a novel HPLC–GPC method was developed based on two serially connected PolarGel-L columns using DMF that contained 5% water, 1% acetic acid and 0.15 M LiBr at 0.7 ml/min and 50 °C. This yielded a single calibration curve for galloyl glucoses (trigalloyl glucose, pentagalloyl glucose), ellagitannins (pedunculagin, vescalagin, punicalagin, oenothein B, gemin A), proanthocyanidins (procyanidin B2, cinnamtannin B1), and several other polyphenols (catechin, epicatechin gallate, epicallocatechin gallate, amentoflavone). These GPC predicted molecular weights represented a considerable advance over previously reported HPLC–GPC methods for underivatized proanthocyanidins.