Separation and characterization of NOM by high-performance liquid chromatography and on-line three-dimensional excitation emission matrix fluorescence detection.

By using high-performance size exclusion chromatography (HPSEC) and reversed-phase high-performance liquid chromatography with on-line three-dimensional excitation emission matrix fluorescence detection, we measured fluorescence properties of natural organic matter (NOM) as a function of molecular size (MS) and polarity. The work was carried out with Suwannee River Fulvic Acid, Aldrich Humic Acid, and a naturally occurring river NOM sample. Significant differences in fluorescence maximum pattern were found as NOM was separated chromatographically based on MS and polarity. There existed a strong relationship between MS, fluorescence pattern, and polarity. Humic-, fulvic-, and protein-like fluorescence fractions had distinct hydrophilic/hydrophobic nature. The results suggest that HPSEC may be better for characterizing major fulvic-like fluorescence and smaller MS fractions but not those having humic- and protein-like fluorescence and larger MS, which may be strongly adsorbed onto the HPSEC column because of their hydrophobic nature. This study has significant implications for further understanding the nature of NOM and its complexation with trace metals.