Fulvic acids from the soil of Peking University (PF) and a Nordic river (NF) were separated into well defined sub-fractions using sequential elution techniques based on eluent polarity. The chemical properties of the fractions including: PF1 and NF1 (eluted by 0.01 M HCl), PF2 and NF2 (eluted by 0.01 M HCl+20% methanol), PF3 and NF3 (eluted by 0.01 M HCl+40% methanol), and PF4 and NF4 (eluted by 100% methanol), were characterized using UV-Visible spectroscopy, elemental analysis and (13)C NMR. The results showed that the UV absorptions of the elution peaks at 280 nm (A280) increased from PF2 to PF4 and NF2 to NF4. No elution peaks were observed for PF1 and NF1. The carbon contents increased from 43.34% to 51.90% and 43.06% to 53.26% while the oxygen contents decreased from 46.39% to 36.76% and 49.76% to 40.03% for PF1-PF4 and NF1-NF4, respectively. As a polarity indicator, the (O+N)/C ratio for PF1-PF4 and NF1-NF4 decreased from 0.88 to 0.62 and 0.89 to 0.58, respectively. The aromatic carbon content increased from PF1 to PF4 and NF1 to NF4, suggesting an increase of the hydrophobicity of these fractions. The polarity was positively related to the ratio of UV absorption at 250 nm and 365 nm (E2/E3), and negatively related to the aromaticity. A high positive relationship between the aromaticity and E2/E3 of fulvic acid fractions was also obtained. The use of an eluent with a decreasing polarity allowed to providing simpler fractions of soil and aquatic fulvic acids.