The reported study describes the separation of humic substances (HS) dissolved in surface waters into hydrophilic and hydrophobic fractions with the SPE method onto StrataX (Phenomenex) columns. The separated fractions were analyzed with the HPLC-C18 (Merck column) method for relative polarity of humic (HA) and fulvic (FA) acids in the samples. Analyses were carried out on samples collected from 7 lakes constituting two cascade systems located in the same catchment of the Ełk River. During HPLC assays, an effect of size exclusion (SE) was observed, especially for the hydrophilic fraction. The HPLC analyses enabled us to demonstrate that in both hydrological systems the hydrophilic fraction contained mainly molecules with molecular weights exceeding 1,000Da and of practically equal relative polarity. Only in system II (five lakes) was the hydrophobic fraction strongly diversified quantitatively as well as in terms of molecular weight and polarity between the samples. Results of analysis show that this system was subject to a strong anthropogenic impact.