Latahco silt-loam humic acid was separated on a preparatory scale by size exclusion chromatography (SEC) on a gravity-fed Sepharose column. Four fractions from this separation were collected and further analyzed, along with whole humic acid, by high-performance SEC coupled with a multi-angle light scattering (MALS) detector. A detailed characterization of the radius of gyration across the elution profile of each fraction by MALS demonstrated that the preparative column generally separated the humic acid according to differences in molecular size. Furthermore, a comparison of elution profiles obtained on the whole humic acid with UV and refractive index detectors revealed the presence of a non-aromatic low molecular weight component.