Evaluation of immobilized metal affinity chromatography (IMAC) for isolation and recovery of strong copper-complexing ligands from marine waters

The speciation of dissolved copper in surface seawater is dominated by complexes with at least two classes of organic ligands, L-1 (the stronger) and L-2 (the weaker). However, because these ligands have never been isolated, recovered, and characterized, their nature and identities are unknown. We have taken a combined approach utilizing immobilized metal affinity chromatography (IMAC) for isolation, and copper titration monitored by differential pulse anodic stripping voltammetry (DPASV) at a thin mercury film, rotating glassy carbon disk electrode for detection of these ligands in marine waters and in chromatographic fractions. We have found that, using the IMAC protocol reported by A.S. Gordon (Isolation of compounds with affinity for copper from seawater using immobilized copper ion affinity chromatography, Mar. Chem. 38 (1992) 1-12), we can remove the majority of L-1 and L-2 from Chesapeake Bay and Atlantic coastal water samples. Once isolated, similar to 67% of the L-1-class ligands bound to the IMAC column could be recovered by elution with 5 mM glycine buffer, as monitored by DPASV copper titration of the column eluent. This recovery accounted for similar to 47% of the original amount of L-1 in the sample and resulted in a 5.4-fold concentration factor (from 7 to 38 nM) of the ligands from the seawater sample into glycine eluent. Although this IMAC protocol traps the weaker, L-2 class ligands as well as L-1 class ligands, confirmation of L-2's recovery by elution of the IMAC column is hindered by the presence of glycine in the eluent. We are currently devising procedures to optimize the isolation and improve recovery efficiency of IMAC for these ligands, in order to obtain sufficient quantities for further characterization.