Marine dissolved organic matter (DOM) was separated by reversed-phase (RP) liquid chromatography method and analyzed with fluorescence/absorption detection and Fourier transform ion cyclotron mass spectrometry (FT-ICR-MS). The three key characteristics of the RP method are: (a) The C18-RP column chosen provides enhanced separation when the aqueous phase is 100% buffer-free water, and it does not degrade over time; (b) the water eluent adjusted to pH 7 significantly improves the resolution of water soluble compounds; (c) the initial flow maintained at low levels improves the separation of polar compounds. In samples, containing "fresh" DOM, specific peaks were detected, which were absent in "old" DOM samples. The combination with size exclusion chromatography (SEC) also demonstrated the relation between polarity and molecular size of DOM. FT-ICR-MS was applied to evaluate the quality of separation on a molecular scale demonstrating that physico-chemical characteristics of DOM can be related to molecular formulas. Sample extracts were separated into 4 preparative fractions, and a large suite of the identified molecular formulas only occurred in specific fractions. This is an important basis for the application of further analytical techniques in order to perform a more target-oriented analysis aiming at the determination of source and process biomarkers for DOM. © 2008 Elsevier B.V.