Argentation chromatography has been developed for the analysis and separation of asphaltenes. The separation is based on the interaction between the silver ions (Ag+) bonded to a silica gel surface and the pi-systems of the polycondensed aromatic compounds, where the interactions can be different when heteroatoms (N, O, S) are present in the molecules. Online coupling of argentation chromatography and high-resolution mass spectrometry allows fast and detailed analysis of the separation of crude oil asphaltenes. Highly condensed aromatic compounds possessing a high degree of unsaturation can be successfully separated using a mixture of toluene and chloroform (7:3 v/v) as mobile phase and dimethyl sulfoxide as competitive ligand for elution of asphaltene compounds with double bond equivalents (DBE) of up to 40. Making use of the different structural features of the components, it is possible to separate isomeric compounds that interact with different strengths with the stationary phase. The composition of mobile phase plays a significant role in separation efficiency.