Resolving the chemical heterogeneity of natural organic matter: new insights from comprehensive two-dimensional liquid chromatography.

For the purpose of resolving the chemical heterogeneity of natural organic matter (NOM), comprehensive two-dimensional liquid chromatography (LC×LC) was employed for the first time to map the hydrophobicity versus molecular weight (MW) distribution of two well-known complex organic mixtures: Suwannee River Fulvic Acids (SR-FA) and Pony Lake Fulvic Acids (PL-FA). Two methods have been developed using either a conventional reversed-phase (RP) silica column or a mixed-mode hydrophilic interaction column operating under aqueous RP mode in the first dimension, and a size-exclusion column in the second dimension. The LC×LC fractions were screened on-line by UV at 254 nm, molecular fluorescence at excitation/emission wavelengths (λ(Exc)/λ(Em)) of 240/450 nm, and by evaporative light scattering. The MW distributions of these two NOM samples were further characterized by number (Mn) and weight (Mw) average MW, and by polydispersity (Mw/Mn). Findings suggest that the combination of two independent separation mechanisms is promising in extend the range of NOM separation. For the cases where NOM separation was accomplished, smaller Mw group fractions seem to be related to a more hydrophobic nature. Regardless of the detection method, the complete range of MW distribution provided by both comprehensive LC×LC methods was found to be lower than those reported in the literature.