The use of a 4.6 x 250 mm, 5 mu m cyanopropyl column is effective for the liquid chromatography (LC) separation of asphaltenes with sequential ultraviolet (UV) and florescence detection. The mobile-phase composition is an optimized gradient from acetonitrile (MeCN) and water to N-methyl-2-pyrrolidone (NMP) and tetrahydrofuran (THF). A low flow rate of 0.5 mL min(-1) is used to maintain lower operating pressure to minimize aggregate formation. Using a 0.02 g L-1 asphaltene sample for preliminary optimization, three peaks, with two partially resolved, are evident in the fluorescence chromatogram. The UV chromatogram revealed an extra weakly retained peak, suggesting aggregates that quench fluorescence. Aggregation of asphaltenes increases with time up to about 10 h and is dependent upon the choice of sample solvent. On the basis of the reversed-phase mobile-phase gradient, the relative polarity of the peaks from least to most retained can be estimated over the polarity index (P') range from about 6.3-4.3 on a scale of 0.1 for hexane (least polar) to 10.6 for water (most polar). The sample concentration is increased to 1 g L-1 for separation and collection of 12 fractions. Selected fractions are subjected to characterization using atmospheric pressure chemical ionization mass spectrometry (APCI-MS) using a linear quadrupole ion trap (LQIT). The variation of the molecular-weight distribution of the asphaltenes for the 12 fractions is fairly constant, indicating that the retention mechanism is not controlled by size exclusion but likely a partitioning/adsorption mechanism.