Development of on-line comprehensive two-dimensional liquid chromatography method for the separation of biomass compounds.

•RPLC×RPLC was successfully used to separate the aqueous phase of a partially dehydroxygenated bio-oil.•A limited set of representative compounds allowed to find suitable 2D-conditions.•The degree of orthogonality between Hypercarb and silica-based columns was found to be good.•The effective practical peak capacity reached almost 2000 by using HT-UHPLC in the second dimension.