Combined HPLC/HPSEC study of Suwannee River Fulvic Acid adsorptive fractionation on α-aluminum oxide

Abstract

A novel liquid chromatographic (LC) method with repeated injections of Suwannee River Fulvic Acid (SRFA) was used to investigate its adsorptive fractionation by synthetic α-Al2O3. Eluent (i.e., non-retained) SRFA for each injection was monitored by two ultraviolet (UV) absorbance detection channels (300 and 365nm) and one fluorescence detection channel (λex=350nm, λem=450nm). Preferential adsorption of SRFA constituents was revealed by the different responses of the three LC detection channels. Samples of non-retained SRFA from injections of three independent replicate experiments were collected and aggregated for subsequent analysis by steady state ultraviolet–visible (UV/vis) absorption spectrometry and size exclusion chromatography (SEC). The ratio of absorbance at 254 and 204nm, a surrogate for specific UV absorbance at 254nm, increased with increasing injection number for the non-retained SRFA, indicating the preferential adsorption of SRFA constituents containing aromatic moieties. SEC analysis confirmed the preferential adsorption of higher molecular weight (MW) SRFA constituents as the non-adsorbed SRFA fractions increased in MW across the series of injections. The SEC results also suggested that certain SRFA constituents in the ca. 2–5kDa MW range adsorbed in early injections were displaced by higher MW species (ca. 5–10kDa) in later injections.