14 15 On-line comprehensive Reversed Phase Liquid Chromatography x Supercritical Fluid 16 Chromatography (RPLC x SFC) was investigated for the separation of complex 17 samples of neutral compounds. The presented approach aimed at overcoming the 18 constraints involved by such a coupling. The search for suitable conditions (stationary 19 phases, injection solvent, injection volume, design of interface) are discussed with a 20 view of ensuring a good transfer of the compounds between both dimensions, thereby 21 allowing high effective peak capacity in the second dimension. Instrumental aspects 22 that are of prime importance in on-line 2D separations, were also tackled (dwell 23 volume, extra column volume and detection). After extensive preliminary studies, an 24 on-line RPLCxSFC separation of a bio-oil aqueous extract was carried out and 25 compared to an on-line RPLCxRPLC separation of the same sample in terms of 26 orthogonality, peak capacity and sensitivity. Both separations were achieved in 100 27 min. For this sample and in these optimized conditions, it is shown that RPLCxSFC 28 can generate a slight higher peak capacity than RPLCxRPLC (620 vs 560). Such a 29 result is essentially due to the high degree of orthogonality between RPLC and SFC 30 which may compensate for lesser peak efficiency with SFC as second dimension. 31 Finally, in the light of the current limitations of SFC instrumentation for on-line 2D 32 analyses, RPLCxSFC appears to be a promising alternative to RPLCxRPLC for the 33 separation of complex samples of neutral compounds. 34 35 36 37 Keywords 38 Supercritical fluid chromatography (SFC); RPLC x SFC; Comprehensive two-39 dimensional chromatography; Biomass by-products 40