Characterization of Cu(II)-binding ligands from the Chesapeake Bay using high-performance size-exclusion chromatography and mass spectrometry

The speciation of Cu(II) in marine waters is dominated by organic ligands, which have resisted detailed chemical characterization. In this work we have used immobilized-metal affinity chromatography (IMAC) to isolate Cu(II)-binding ligands from the Chesapeake Bay. We have then used high-performance size-exclusion chromatography (HPSEC) and mass spectrometry (MS) to gather information about the size distributions, molecular weights, and chemical functionality of the ligands isolated by IMAC. Results show that weaker-binding ligands have molecular weights that range from about 230 up to >20,000 Da. A portion of these weaker-binding ligands has molecular weight distributions that are consistent with humic substances. The molecular weight distribution of stronger-binding ligands is significantly more narrow with molecular weight values that are less than 1600 Da. Both HPSEC and MS show that the most abundant stronger-binding ligands have molecular weights around 270 Da. Furthermore, mass spectral analysis allows some empirical molecular formulas to be postulated for several ligands. These empirical formulas show that the ligands are abundant in nitrogen, oxygen, and sulfur functionality. In addition, consistency between MS data and data from HPSEC when peptides are used for the calibration combined with the low molecular weights and prevalence of nitrogen, oxygen, and sulfur functionality suggest that the stronger-binding ligands may have been produced directly by organisms in the water. It is not clear at this point, however, the degree to which the molecular information we have gathered represents the majority of the Cu(II)-binding ligands at our sampling site. Nonetheless, combining IMAC, HPSEC, and MS seems to be a promising approach for characterizing Cu(II)-binding ligands in natural waters.