| Humic substances (HS) are polyelectrolytic macromolecules derived from organic components in soil and water. The critical roles HS play in the environment make it necessary to improve their characterization, which is complicated by their inherently heterogeneous nature and polyfunctionality. A robust characterization method requires sensitivity to minor differences in structural composition between samples. The goal of this dissertation research is to investigate the use of chromatographic and electrophoretic techniques with absorption and fluorescence detection to provide a detailed profile of HS as a function of size.;The first project utilizes a high-performance size exclusion (HPSEC) column, G3000PW TSKgel, to characterize by MW using poly (styrene sulfonates) as calibration size standards. The goal was to establish MW values for several IHSS HS. Separation of HS using the TSKgel column generated MW values, ranging from 2,500--10,000 Da, which are in the range reported in the literature (800--20,000 Da). Structural subunits of HS, primarily aromatic and aliphatic carboxyl, hydroxyl and phenolic groups, were tested to determine the possible role of secondary interactions, including adsorption and ion exclusion, which may affect separation. The calculated MW values for the structural subunits indicated that both secondary interactions must be considered when evaluating MWs obtained with the TSKgel column.;The second project utilized the charged nature and intrinsic fluorescence of HS for characterization by capillary gel electrophoresis with on-the-fly fluorescence lifetime detection (CGE-OFLD). Recent studies in our group found that HS exhibits 2--3 lifetime components across the broad, humic hump in capillary zone electrophoresis (CZE). To complement these findings, this project added a poly (ethylene glycol) gel to the mobile phase and used a poly (vinyl alcohol) coated capillary, in order to decrease the mobilities of the HS and add a sieving mechanism to separation achieved with CZE. The results showed that fluorescence lifetime decreases with increasing size of the HS molecule. In addition, all HS exhibited a bi-exponential decay across the humic peak. Lifetime-resolved electropherograms of the lifetime components varied according to source (soil, peat, and aquatic). This suggests that OFLD is sensitive to the MW distribution of HS in the CGE experiment. |
