In this study, the potential of using off-line or online two dimensional liquid chromatography (2D LC) separations of plant extracts was examined.A new approach to high speed, online isocratic elution comprehensive 2D LC based on the use of hydrophilic interaction liquid chromatography (HILIC) chromatography in the first dimension, and a fast second dimension utilizing high temperature and Hypercarb porous carbon stationary phase is described. Entirely conventional isocratic elution instrumentation and columns were assembled in a system which could separate the complex mixture of compounds extracted from rare Serbian plants. The second dimension column was used at high flow rate in isocratic mode and a high column temperature of 110°C. Fractions eluted from the first dimension HILIC column were subjected to the second dimension either online (using an interface) or off-line. The online HILIC-Hypercarb combination demonstrated large peak capacities as compared to other modes.