Molecular size distribution and spectroscopic properties of aquatic humic substances

The number- and weight-averaged molecular weights of different isolated humic fractions and unfractionated organic matter from natural water samples were measured using high-performance size-exclusion chromatography (HPSEC). The chromatograms obtained, with sodium acetate as the mobile phase on a TSK G3000SW high-speed column, provided illustrative information about the nature of the dissolved organic matter that was not available with phosphate buffer eluents. The calibration of the column system merely with protein standards generated misleading information about the molecular weight and size distribution of humic solutes. Instead, the present results support the utilization of polystyrene-sulphonates (PSS) either alone or together with certain other model compounds for the calibration of HPSEC in studying molecular weights of humic solutes. The data obtained with the HPSEC system indicate that the average molecular weight of humic solutes is distinctly lower than is generally assumed. Besides, aquatic humic solutes also contain some very large-sized though minor constituents, leading to a fairly polydisperse mixture. Number-averaged molecular weights obtained using the HPSEC system were 4-5-fold greater than those from vapour-pressure osmometry. Bulk spectroscopic properties such as molar absorptivity at 280 nm and the quotient, E(2)/E(3), correlated strongly with the total aromaticity and averaged molecular weights of all the humic solutes. This observation suggests that bulk spectroscopic properties can be applied, as a first approximation, for estimating the size of humic solutes and their aromaticity in natural surface waters.