The acidic components of Athabasca bitumen interfacial material (IM) were isolated and subsequently fractionated based on hydrophobicity by a modified aminopropyl silica (MAPS), method to determine whether low-molecular-weight IM acids are preferentially ionized in negative-ion electrospray ionization (ESI (-)) and, thus, bias the compositional information obtained by direct infusion (dilute and shoot) mass spectral analysis. Characterization by negative-ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) revealed that MAPS fractionation of IM acids extends the detection of high-m/z (>600-Da) IM compounds by 2-fold, yields an approximate 10-fold increase in the number of assigned formulas, and exposes a continuum of acidic species that includes the first definitive identification of doubly charged acids in interfacial material. Comparison of the heteroatom contents of singly and doubly charged O-chi species, combined with the acid-targeted extraction procedure, strongly suggests that the chemical functionalities are similar for the two ion types and are largely composed of mono- and, dicarboxylic acids. Excitation emission Matrix spectroscopy, (EEMS) revealed that the most hydrophobic IM fractions approach the size and aromaticity of bitumen asphaltenes, but despite the increase in hydrophobicity, these asphaltene-like acids form the tightest emulsions, as revealed in simple bottle tests. Thus, the most surface active material from Athabasca bitumen comprises low-molecular-weight, acidic, resin-like species, as well as larger (higher-moleeular-weight) acidic asphaltene-like compounds.