Separation and chemical analysis was investigated using bitumen samples from Athabasca oil sand in Alberta. Fractionation according to solubility and polarity has been used to separate bitumen into its fractions. The solvent de-asphaltening was performed by n-pentane solvent (solubility fractionation), and the polarity fractionation using Fuller’s earth allows maltene to separate into SARA components (saturates, aromatics, resins and asphaltenes). The SARA components are analyzed comprehensively using elemental analysis (EA), Fourier-transformed infrared (FTIR), ultraviolet-visible spectroscopy (UV-vis), high performance chromatography (HPLC) and thermogravimetric analysis (TGA). EA (C, H, N, S), heavy metals (Ni, V) concentrations, FT-IR and UV-vis tests provided the explanation of chemical composition. From IR spectra, maltene and saturates/aromatics (sat/aro) contained more aliphatic compounds than resin or asphaltene. Also, IR spectrum of sat/aro was similar to crude oil and VGO (vacuum gas oil). Different UV signal data clearly indicates the contribution of aromatic constituents in the fractions. Using optimized analysis conditions of HPLC, we successfully separated the peaks for bitumen and its fractions. The characteristic peak pattern of SARA (saturates, aromatics, resins, asphaltenes) fractions was observed, and also the peak pattern of sat/aro was similar to that of crude oil and VGO. However, TGA results revealed that thermal behavior for sat/aro was similar to that of crude oil but different from that of VGO. Also, from the comparison between decomposition temperature of TGA and boiling point, their correspondence was found.