Why proanthocyanidins elute at increasing order of molecular masses when analysed by normal phase high performance...

Although it is widely known that proanthocyanidins elute at an increasing order of molecular masses when analysed by normal phase high performance liquid chromatography (NP-HPLC), there is no a consistent explanation of the mechanisms of their separation until now. Therefore, the aim of the present study was to give a reliable response to this question, using a complex procyanidin-rich grape seed extract. For this, an off-column static simulation of extract injection and a fragmented-column dynamic procyanidin location tests were studied to show their precipitation in an aprotic solvent, besides another off-column static simulation and multiple contact dynamic solubilisation tests to confirm procyanidin redissolution in an aprotic/protic solvent system. The results showed that separation of procyanidins in the aprotic/protic solvent system of Diol-NP-HPLC was governed by precipitation/redissolution mechanism, that could be extended to all known plant proanthocyanidin homopolymers, including hydrolysable tannins, if they are able to accomplish this condition. However, separation of monomer species, namely catechins and some hydroxybenzoic acids, was based on classic adsorption/partition mechanism. Other factors, such as analyte solubility, chromatographic conditions and sample preparation, that affect the viability of proanthocyanidin analysis by NP-HPLC were stand out and guidelines for its durable and reproducible use were defined.