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 metal affinity chromatography (IMAC). Seasonal and diurnal variation of copper ligand concentrations as measured by IMAC peak areas were determined. Apparent molecular weight and UV-visible spectra of IMAC-isolated ligands were characterized. The effects of sample pH, flow rate, volume and storage (4 °C) on ligand recovery were determined. Removal of ligands of the L1 and L2 class from water samples by IMAC was measured using differential pulse anodic stripping voltammetry (DPASV). Removal of IMAC ligands from acidified water samples by XAD resin was evaluated in order to determine whether IMAC-isolated ligands are humic substances. Considerable variation was observed in IMAC peak areas over a 12-month period. Maximum peak areas of the high-affinity ligand (peak B) were observed in late summer corresponding to maxima in picoplankton abundances in the vicinity of the sample site. Diurnal variation was minimal. Molecular filtration indicated that IMAC-isolated ligands were primarily of low (< 10,000 Da) molecular weight. Spectral analysis of ligands contained in IMAC peaks showed significant heterogeneity 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 IMAC fractions. Removal of humic substances from water samples using XAD prior to IMAC reduced IMAC peak areas ˜ 50%, indicating that 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 temporal variation, are primarily low molecular weight and contain humic substances. Temporal variation observed for the high-affinity ligand class suggests that the ligands may be a product of picoplankton in the water column. The data suggest that IMAC may be a useful method for preparative isolation of high-affinity copper ligands of the L1 and L2 class.