Comprehensive two-dimensional liquid chromatography (LC£LC) coupled with a diode array detector (DAD) has been used for the first time to investigate chemical heterogeneity and map the hydrophobicity versus molecular weight distribution of the most hydrophobic acid fractions in water-soluble organic matter (WSOM) from fine atmospheric aerosols collected over different seasons at a urban location. The use of LC£LC-DAD, combining the use of two independent separation mechanisms (per aqueous liquid chromatography [PALC] versus sizeexclusion chromatography [SEC]), has shown a great potential for unraveling and resolving the heterogeneity of WSOM, further providing a deeper insight into how size-distinguished aerosol WSOM fractions differ in hydrophobicity during different seasonal events. The combination of PALC£SEC-DAD data with an already proposed data treatment procedure revealed that the WSOM samples collected during warm seasons are enriched in aliphatic structures, while those collected during colder seasons exhibit a higher degree of structures with conjugation of p-bonds (e.g., aromatics) alongside aliphatic structures.