Effect of detector wavelength on the determination of the molecular weightof humic substances by high-pressure size exclusion chromatography

High-pressure size exclusion chromatography (HPSEC) has proven to be an eff ective method for determining the molecular weights (MW) of humic substance s (HS) from a variety of aquatic and terrestrial environments. HPSEC system s often use a variable wavelength UV-vis detector, which detects the analyt es based upon their chromophoric composition. HS contain a range of moietie s with chromophores having unique molar absorptivities (for a given wavelen gth), and the calculated MW may be dependent upon the wavelength chosen for the analysis. As a consequence, the choice of wavelength becomes an import ant parameter for the reliable determination of MW by HPSEC. The effect of UV-vis detector wavelength on the determination of the MW distribution of s elected humic and fulvic acids by HPSEC is examined in this paper. For the HS examined, both the number (M-n) and weight average (M-w) MW increased wi th increasing wavelength. The relative increase in MW was most pronounced f or Lake Fryxell fulvic acid, with a 63 and 21% increase in M-n and M-w, res pectively, between 220 and 380 nm. The increases observed for Suwannee Rive r humic and fulvic acids were less pronounced, M-n was more sensitive to ch anges in detector wavelength than M-w, and as a result the target HS appear ed to be less polydisperse at higher wavelengths. Within the range of wavel engths commonly used for the determination of MW of HS by HPSEC (i.e., 220- 280 nm), the magnitude of the increases in MW was not significant compared to variability in MW that results from changes to other operational paramet ers in HPSEC. (C) 2000 Elsevier Science Ltd. All rights reserved.