Humic acids (HAs) play important roles for the fate of metal ions in the environment. Most chemical speciation models involving HAs assume heterogeneous metal ion binding. However, these models also assume that the binding affinities of metal ions with HAs are the same regardless of the molecular weight (MW) ranges of the HAs involved. Here, we develop new polyacrylamide gel electrophoresis (PAGE) techniques to investigate the MW distributions of HAs with strongly complexed Cu 2+ ions. By combining contaminant metal-free and high-resolution PAGE for HAs, this work was able to provide accurate MW distributions for the complexed metal ions. The MW distribution of Cu 2+ binding ability per quantity of HA indicates that strong metal-binding moieties in HAs are heterogeneous in terms of MW. Coupling of the PAGE techniques with UV−vis and excitation−emission matrix (EEM) spectrometry-parallel factor analysis (PARAFAC) methods revealed new insights into kinetically inert interactions between HAs and Cu 2+ ions. By this method, we found that the protein-like fluorescence components in the high-and low-MW regions cooperatively responded through Cu 2+ binding. Thus, the advanced gel electrophoresis techniques developed herein are able to shed new light on the heterogeneity of metal binding affinities of HAs in terms of MW.
A novel combination of CE-based separation techniques was used for the precise fractionation of ionic compounds from impurities. The combination of on-capillary concentration and separation using transient isotachophoresis, with multiple injections and a two-point detection system provided higher efficiency, and accuracy at a microliter-scale injection volume, than when CE was individually used for purification. In this paper, we present successful applications of the CE fractionation techniques for the purification of fluorescein, fluorescein-4-isothiocyanate, two fluorescent metal ion probes, and a fluorescein-modified DNA aptamer. The purity of the isolated fluorescent probes ranged from 95 to 99%. Such high purity could not be achieved using chromatographic purification techniques. With relatively low dilution factors of 6-9, the purified probe solutions were practical for use as purified stock solutions. In addition, the fluorescein-modified DNA aptamer purified by our method was successfully used in a thrombin binding assay. The method developed was useful for the purification of anionic fluorescent reagents to be of ultratrace analytical grade for use with CE-LIF.
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