Humic substances (HS) is considered a critical factor in assessing the fate, the mobility, and the toxicity of metals in aqueous systems under a fixed solution chemistry of pH, dissolved organic carbon, competing ions, and ionic strength. Due to the strong merits of fast, high sensitive, and cost-effective measurements and fluorescence quenching phenomenon upon the interaction with metals, fluorescence quenching method has been widely used to probe the binding affinity of HS or dissolved organic matter (DOM). Two dimensional correlation spectroscopy (2D-COS) is a powerful mathematical tool to offer a clear picture of spectral changes of a complex mixture upon the external perturbations over a wide range of spectral variables. To our best knowledge, no studies was previously conducted to explore the molecular heterogeneity with respect to metal binding by using 2D-COS combined with SEC, which is called here two-dimensional size exclusion chromatogram (2D-Co-SEC). The objectives of this study were (1) to test the feasibility of 2D-Co-SEC for characterizing metal-HS binding properties, and (2), using the new technique, to obtain a heterogeneous and continuous distribution of copper binding sites within a bulk HS with respect to the molecular size.
In order to ensure the feasibility of the 2D-Co-SEC for assessing the molecular weight distribution with respect to copper binding, neither mass loss nor no disruption in the original HS structures occur for both the dissolved and the complex phases along the passage through the SEC column. To test the two possibilities, we compared SEC chromatograms using UV detector with increasing copper ions. Combined results confirmed that no substantial loss of DOM by metal complexation occurred within the SEC column, suggesting that the fluorescence quenching phenomenon observed from the SEC chromatograms could be interpreted by copper binding.
Fluorescence quenching was observed upon the addition of copper over a wide range of MW values in two types of humic acid, suggesting that a range and distribution of molecular sized molecules are involved in copper binding. Although it is clear that the spectral quenching occurred over the ranges of MW values or a range of the MW distribution, such one-dimensional data have limitations for fully capturing the uneven distribution of the copper binding properties with respect to HS structures. To overcome the constraints and to enhance the spectral resolution, further analysis of the correlations between one-dimensional signals is needed using advanced tools such as 2D-COS.
A series of the SEC chromatograms of HS sequentially changed with varying copper concentrations were incorporated into 2D-COS. Based on Noda’s rule, these results suggest that the copper binding affinity of HS tends to be stronger with increasing molecular size except for the largest size group with the representative MW value of ~15800 Da. The order of the calculated constants was very consistent with the interpretation from the 2D-Co-SEC maps. We compared the heterogeneous distribution of copper binding affinity over a range of MW values obtained from those of the SEC chromatograms versus the five UF size fractions. As expected, the binding constants from the UF fractions exhibited a much shorter range than those obtained from the SEC chromatograms. Our proposed 2D-CoSEC method would provide a high resolution in separating metal-binding characteristics of HS over a wide range of molecular sizes, and it can give a clear picture of the size-dependent distribution of metal-HS interactions.