HPSEC as a prepative fractionation technique for studies of natural organic matter (NOM)

High pressure size exclusion chromatography has been investigated as a preparative fractionation technique for studies of natural organic matter. Five different experiments were conducted to examine the effect of gel-solute interactions in the selected high pressure size exclusion chromatography system, and to evaluate the reproducibility of the method: (1) different flow rates, (2) different sample concentrations, (3) reinjection of collected fractions, (4) addition of acetonitrile to the mobile phase, and (5) reinjection of hydrophobic and hydrophilic sub fractions. The results show that gel-solute interactions are a minor problem, and that the selected high pressure size exclusion chromatography system separates the natural organic matter molecules mainly on the basis of molecular size. The optical properties (ultraviolet/ visible and fluorescence) vary systematically between the collected fractions. The main distinguishing features are relatively stronger absorbancies at shorter (<290 nm) wavelengths for smaller molecules, strong specific ultraviolet/ visible absorbancy for natural organic matter molecules of intermediate molecular mass, and very low fluorescence of larger molecules.