The analysis of a Maya asphaltene using size-exclusion chromatography (SEC) and 1-methyl-2-pyrrolidinone (NMP), tetrahydrofuran (THF), and toluene as the eluent (separately used) showed radically different chromatograms. The SEC of fossil fuel hydrocarbons, e.g., asphaltenes, using NMP has shown a two-peak structure, which has been the source of debate, whether the peak excluded from the column porosity (appearing at the shorter retention times) contained asphaltenic aggregates or very large molecular masses of thousands of mass units. The SEC of asphaltenes using THF and toluene showed retained peaks at the longer retention times. In this work, atomic force microscopy (AFM), a technique capable of reliable and accurate detection of the interaction forces between various molecules and in different media, was exploited to investigate the interaction forces between polystyrene (PS) particles (SEC column packing) and asphaltenes in air and in NMP. AFM showed a strong interaction between the PS particles and asphaltenes, the outcome of which can be the aggregation of asphaltenes in the SEC columns. The use of solvents, such as toluene, NMP, and THF, which either damage the column packing or do not dissolve asphaltenes completely, the interaction between asphaltenes, and the SEC column packing and aggregation of asphaltenes in the SEC columns are the major limitations of SEC in analyzing asphaltenes. Furthermore, PSs, currently used to calibrate SEC columns, are significantly different from asphaltenes in their structure and solubility behavior. In addition, the quantitative results of the SEC are considerably different from the reports from other techniques, e.g., LD, MALDI, FT-ICR MS, and molecular diffusivity methods. Thus, SEC results cannot be used quantitatively to determine mass distributions of asphaltenes.