An off-line multidimensional method involving liquid chromatography combined with supercritical fluid chromatography was developed for the characterization of the wastewater of hydrothermal liquefaction of microalgae Chlorella sorokiniana. The first dimension consisted of a phenyl hexyl column operated in reversed-phase mode, whereas the second dimension was performed on a diol stationary phase. Optimization of the kinetic parameters of the first and second dimensions were performed, taking into account the fraction collection system. The beneficial effect of working at high flow rate in both dimensions, as well as the need to work with short columns (50 mm) in the second dimension was evidenced. Injection volume was also optimized in both dimensions. The first dimension benefited from on-column focusing, while in the second dimension, untreated water-rich fractions could be injected without peak deformation. The performances of offline LCxSFC were compared to LC-HRMS, SFC-HRMS and LCxLC-HRMS for the analysis of the wastewater. Despite a long analysis time of 3.3h, the off-line separation coupled to high-resolution mass spectrometry exhibited a very large orthogonality with 75 % occupation rate of the separation space, reaching an effective peak capacity of 1050. While other evaluated techniques were faster, one-dimensional techniques failed to separate the numerous isomers while LCxLC exhibited lower orthogonality (45% occupation rate).