Different soil extracted humic acids as well as a commercial humic acid sodium salt were fractionated by HPLC. An almost complete recovery could be achieved for the dissolved material. All humic samples show a typical chromatogram of at least five fractions. The separation of humic substances is influenced by an altered hydrophobicity due to changes in the tertiary structure. Three fractions were further investigated by Diffuse Reflectance Infrared Fourier Transform (DRIFT) spectroscopy and by HPLC/HPLC separation. DRIFT spectroscopy provided data on the primary structure, whereas HPLC/HPLC results gave insight into the secondary and tertiary structure and their changes.