COPPER LIGANDS ISOLATED FROM ESTUARINE WATER BY IMMOBILIZED METAL AFFINITY-CHROMATOGRAPHY - TEMPORAL VARIABILITY AND PARTIAL CHARACTERIZATION

Copper ligands were isolated from estuarine water using immobilized me tal affinity chromatography (IMAC). Seasonal and diurnal variation of copper ligand concentrations as measured by IMAC peak areas were deter mined. Apparent molecular weight and UV-visible spectra of IMAC-isolat ed ligands were characterized. The effects of sample pH, flow rate, vo lume and storage (4 degrees C) on ligand recovery were determined. Rem oval of ligands of the L(1) and L(2) class from water samples by IMAC was measured using differential pulse anodic stripping voltammetry (DP ASV). Removal of IMAC ligands from acidified water samples by XAD resi n was evaluated in order to determine whether IMAC-isolated ligands ar e humic substances. Considerable variation was observed in IMAC peak a reas over a 12-month period. Maximum peak areas of the high-affinity l igand (peak B) were observed in late summer corresponding to maxima in picoplankton abundances in the vicinity of the sample site. Diurnal v ariation was minimal. Molecular filtration indicated that IMAC-isolate d ligands were primarily of low (<10,000 Da) molecular weight. Spectra l analysis of ligands contained in IMAC peaks showed significant heter ogeneity on different sampling dates. Characteristic spectra exhibited absorbance peaks around 280 nm suggestive of the presence of proteins or peptides containing aromatic amino acids. No DNA was detected in I MAC fractions. Removal of humic substances from water samples using XA D prior to IMAC reduced IMAC peak areas similar to 50%, indicating tha t a significant portion of IMAC-isolated material was humic substances . The results show that copper ligands isolated and recovered by IMAC are a heterogeneous group of compounds that exhibit significant tempor al variation, are primarily low molecular weight and contain humic sub stances. Temporal variation observed for the high-affinity ligand clas s suggests that the ligands may be a product of picoplankton in the wa ter column. The data suggest that IMAC may be a useful method for prep arative isolation of high-affinity copper ligands of the L(1) and L(2) class.