Two-dimensional reversed-phase liquid chromatography using two monolithic silica C18 columns and different mobile phase modifiers in the two dimensions.

Comprehensive two-dimensional (2D) HPLC in the reversed-phase liquid chromatography (RPLC) mode using C18 silica monolith columns at first dimension (1st-D) (10 cm x 4.6mm I.D.) and second dimension (2nd-D) (5 cm x 4.6mm I.D.) was carried out successfully. A mixture of water and tetrahydrofuran (THF) was used as a mobile phase in the 1st-D separation, and a mixture of water and methanol (CH3OH) in the 2nd-D separation. Sample fractions from 1st-D column were directly loaded into an injection loop of the 2nd-D HPLC equipped with two injector valves for one column. The fractionation time at the 1st-D that was equal to the separation time at the 2nd-D was 45 or 60s. Total peak capacity up to 900 was obtained in about 60 min for the isocratic mode separation of aromatic compounds in this system. Gradient elution mode applied to both 1st-D and 2nd-D separations resulted in shorter separation time and better separation efficiencies than the isocratic mode. It was demonstrated that 2D-HPLC systems employing popular C18 stationary phases with different organic modifiers in mobile phases for each dimension could produce large peak capacity. The different selectivities were provided by the difference in polar interactions between a solute and the organic modifier existing in the stationary phase.