Fluorescence spectroscopy of high performance liquid chromatography fractionated marine and terrestrial organic materials

Marine dissolved organic material (“marine DOM”, Mediterranean Sea) and a well characterized soil fulvic acid (“SFA”, Conway) were extracted using tC18 Sep-Pak cartridges. The extracts were characterized by fluorescence spectroscopy before and after reverse phase high performance liquid chromatographic fractionation (RP-HPLC). Based on soil fulvic acid, the tC18 Sep-Pak extraction procedure led to a loss of 65% of the fluorophores at λex 350 nm, and 34% of those at λex 450 nm, yet with no alteration in wavelength maxima; similar behavior was obtained for the marine DOM. Conventional fluorescence emission showed that the SFA fluorescence is 2.5 times higher than that of the marine DOM at λex/λem 350/452 nm and 11 times higher at λex/λem 450/522 nm. The higher fluorescence emission of the SFA in relation to the marine DOM can be attributed to compounds of high polarity. A blue shift was obtained when comparing the emission maximum of marine DOM (λem 437 nm) to that of SFA (λem 452 nm) at a fixed excitation of 350 nm, whereas no significant difference was obtained when excitation was fixed at 450 nm. Several distinct classes of fluorophores were detected in both marine DOM and SFA by the synchronous excitation technique: λex/λem 317/335 nm and 486/504 nm are present in the SFA, and λex/λem 352/370 nm and 566/584 nm in the marine DOM.