R.D. Evans

High-performance liquid chromatographic fractionation and characterization of fulvic acid

High-performance immobilized metal ion affinity chromatography (HP-IMAC) was used to fractionate humic substances (HS) based on their affinity for the immobilized copper(II) ion using acidic and glycine eluents. The work was carried out with two naturally occurring aqueous fulvic acids and commercially available Suwannee River fulvic acid. The IMAC-fractionated HS were then characterized by reversed-phase high-performance liquid chromatography (RP-HPLC) and size exclusion chromatography. The results showed that the affinity HS fraction eluted first using an acidic pH = 2 eluent exhibited a relatively high hydrophilic character, whereas the fraction eluted later using a glycine eluent exhibited both a higher hydrophobic character and larger molecular size. On the other hand, the HS fraction with no affinity for the immobilized copper had low molecular size. The affinity of the HS fraction for copper(II) increased with increasing molecular weight. Based on the composite results of three different HS, it is evident that strong relationships exist between affinity, molecular weight, and hydrophilic/hydrophobic properties during the HP-IMAC fractionation. The results presented here have significance for understanding the nature of chemical interactions at the molecular level between dissolved organic matter and trace metals. IMAC, coupled with other liquid chromatographic strategies, is a promising tool for chemical fractionation and characterization of HS.

Fractionation and characterization of fulvic acid by immobilized metal ion affinity chromatography

Abstract Immobilized metal ion affinity chromatography (IMAC) has been used to separate humic substances (HS) based on their affinity for metal ions. The work was carried out with a stream fulvic acid (FA) as a representative HS. The effect of pH and ionic strength on the retention of HS was investigated, and comparison of different metal ions (Cu 2+ , Ni 2+ , Co 2+ and Cd 2+ ) for IMAC of HS was examined. pH dependence of retained HS and retention volume, and salt-promoted complexation were found; these results are similar to those reported for some proteins using IMAC in previous studies. The HS binding capacity and retention volume in four metal ions studied for IMAC were in the order: Cu 2+ >Ni 2+ >Co 2+ >Cd 2+ , which agrees with the Irving–Williams series. The HS retained on the Cu 2+ -IMAC was eluted with eluent of decreasing pH value, and the fractions collected were characterized by UV–VIS and fluorescence spectra, and fluorescence quenching titration. The results show that HS fractions eluted with lower pH eluent have a higher conditional stability constant ( log K Cu – HS ′ ) for copper(II), red-shifted maximum excitation (Ex) and emission (Em) fluorescence wavelengths (shifted towards longer wavelengths), lower fluorescence (Flu)/absorbance (Abs) ratios, spectral slope and E 265 nm /E 365 nm , indicating that the stronger affinity HS fractions may have relatively higher molecular size and a higher proportion of compounds absorbing and fluorescing at longer wavelength. Based on the results obtained, IMAC appears to be a promising tool for chemical separation of HS. The results will help in the understanding of the nature of HS and their metal binding characterization, and in modelling natural environments.