Fractionation of Dissolved Organic Matter on Coupled Reversed-Phase Monolithic Columns and Characterisation Using Reversed-Phase Liquid Chromatography-High Resolution Mass Spectrometry

Eleven Onyx monolithic C₁₈ columns (100 x 3 mm) were connected in series to obtain a high-capacity reversed-phase HPLC column providing 110,000 theoretical plates. The column was used to fractionate a complex mixture of semi-polar and apolar components within marine dissolved organic matter (DOM), isolated using solid-phase extraction (SPE) on poly(styrene–divinylbenzene) cartridges, and Suwannee River natural organic matter, isolated by reverse osmosis. In each case, 15 isolated fractions were further separated using a second-dimension reversed-phase HPLC coupled with high-resolution mass spectrometry. Successful fractionation of the major compositional materials within DOM (i.e. carboxylic-rich alicyclic molecules, CRAM) in order of decreasing polarity was confirmed. Upon formulae assignment, homologous series of compounds were fractionated according to decreasing O/C ratio and increasing H/C ratio. Different compounds with the same exact masses were separated across six fractions, highlighting the immensely complex nature of the material, with the presence of potentially hundreds of molecules for each molecular formula. Both samples showed clear compositional differences not only related to source (seawater or freshwater), but also to the extraction method applied (SPE or reverse osmosis).