Why proanthocyanidins elute at increasing order of molecular masses when analysed by normal phase high performance liquid chromatography? Considerations of use

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 con-sistent 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 ex-tract. 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 re -dissolution 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 tan-nins, 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 re-producible use were defined.(c) 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )

Automated summary

This study provides a reliable answer to the mechanism of proanthocyanidin separation in the nonpolar/polar solvent system of NP-HPLC. The precipitation/redissolution mechanism governs the separation of proanthocyanidins, while the classic adsorption/partition mechanism is responsible for the separation of monomer species. Additionally, the study highlights other factors that affect the analysis of proanthocyanidins by NP-HPLC and provides guidelines for its durable and reproducible use.