Department of Environment and Energy, Sejong University, Seoul 05006, South Korea.
Department of Environment and Energy, Sejong University, Seoul 05006, South Korea.
Environ Pollut. 2017 Aug;227:490-497. doi: 10.1016/j.envpol.2017.04.099. Epub 2017 May 8.
Knowledge of the heterogeneous distribution of humic substances (HS) reactivities along a continuum of molecular weight (MW) is crucial for the systems where the HS MW is subject to change. In this study, two dimensional correlation spectroscopy combined with size exclusion chromatography (2D-CoSEC) was first utilized to obtain a continuous and heterogeneous presence of copper binding characteristics within bulk HS with respect to MW. HS solutions with varying copper concentrations were directly injected into a size exclusion chromatography (SEC) system with Tris-HCl buffer as a mobile phase. Several validation tests confirmed neither structural disruption of HS nor competition effect of the mobile phase used. Similar to batch systems, fluorescence quenching was observed in the chromatograms over a wide range of HS MW. 2D-CoSEC maps of a soil-derived HS (Elliot soil humic acid) showed the greater fluorescence quenching degrees with respect to the apparent MW on the order of 12500 Da > 10600 Da > 7000 Da > 15800 Da. The binding constants calculated based on modified Stern-Volmer equation were consistent with the 2D-CoSEC results. More heterogeneity of copper binding affinities within bulk HS was found for the soil-derived HS versus an aquatic HS. The traditional fluorescence quenching titration method using ultrafiltered HS size fractions failed to delineate detailed distribution of the copper binding characteristics, exhibiting a much shorter range of the binding constants than those obtained from the 2D-CoSEC. Our proposed technique demonstrated a great potential to describe metal binding characteristics of HS at high MW resolution, providing a clear picture of the size-dependent metal-HS interactions.
腐殖质（HS）反应性沿分子量（MW）连续体的异质分布的知识对于 HS MW 发生变化的系统至关重要。在这项研究中，首先利用二维相关光谱结合尺寸排阻色谱（2D-CoSEC）获得了 HS 中铜结合特性随 MW 的连续异质存在。将具有不同铜浓度的 HS 溶液直接注入以 Tris-HCl 缓冲液作为流动相的尺寸排阻色谱（SEC）系统。几项验证测试证实，HS 既没有结构破坏，也没有流动相的竞争作用。与批处理系统类似，在 HS MW 较宽的范围内观察到荧光猝灭在色谱图中。土壤衍生的 HS（Elliot 土壤腐殖酸）的 2D-CoSEC 图谱显示，荧光猝灭程度相对于表观 MW 较大，顺序为 12500 Da>10600 Da>7000 Da>15800 Da。基于改进的 Stern-Volmer 方程计算的结合常数与 2D-CoSEC 结果一致。与水生 HS 相比，土壤衍生的 HS 中 HS 内铜结合亲和力的异质性更大。使用超滤 HS 级分的传统荧光猝灭滴定法无法描绘铜结合特性的详细分布，表现出的结合常数范围比 2D-CoSEC 获得的要短得多。我们提出的技术具有在高 MW 分辨率下描述 HS 金属结合特性的巨大潜力，为尺寸依赖性金属-HS 相互作用提供了清晰的图像。
