Chromatographic Separation of Phenolic Compounds from Extra Virgin Olive Oil: Development and Validation of a New Method Based on a Biphenyl HPLC Column

Three different high performance liquid chromatography columns were accessed for phenolic compounds (PC) separation in the hydrophilic fraction of extra virgin olive oil (EVOO). Two fully porous C-18 bonded silica phases and one partially porous biphenyl column were used. Biphenyl column allowed for an increase of more than 30% in peak capacity (n(c)), higher selectivity () (1.045), and improved retention (k), with a reduction of 22.1% in the retention time. The higher resolution (R-s) was obtained by using the biphenyl column, with a fair separation of oleuropein aglycone isomers (OAI) and a good identification of caffeic acid (CA). Tyrosol (T), hydroxytyrosol (HT), and dihydroxyphenyl glycol (DHPG) were also well separated and identified. Moreover, the method using a biphenyl column was fully validated according to the requirements for new methods. For all parameters, the method applying the biphenyl column proved to be a reliable, accurate, and robust tool for separation, identification, and quantification of the main PCs in EVOOs.