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Theory and application of affinity liquid chromatography and capillary electrophoresis
Abstract
Section 1: Affinity chromatography. A high-performance affinity chromatographic column containing immobilized human serum albumin (HSA) was used for the chiral separation of D- and L-tryptophan and to study the binding of these compounds to HSA. Frontal analysis was used to examine changes in the association equilibrium constant (K$\sb{\rm a}$) and amount of binding sites (m$\sb{\rm L}$) for D- and L-tryptophan on an immobilized HSA column under various elution conditions. Zonal elution was also used to examined the band-broadening due to the slow dissociation of D-and L-tryptophan from this immobilized HSA column. From this data, the dissociation (k$\sb{\rm d}$) and association rate constants (K$\sb{\rm a}$) of these compounds were obtained. The thermodynamic parameters for these binding process were also obtained by using these two methods. It was found the values of K$\sb{\rm a}$, k$\sb{\rm d}$, and k$\sb{\rm a}$ or the number of binding sites on a HSA column can be changed by varying the temperature and mobile phase composition. Section 2: Capillary electrophoresis. The theory and mechanism of chiral separations and solute-ligand binding in capillary electrophoresis based on the use of running buffer additives were examined in this study. Human serum albumin (HSA) and $\alpha$-, $\beta$-cyclodexrtrins were used as model ligands; D/L-tryptophan, R/ S-warfarin and o-,m-,p-nitrophenols were used as test analytes to be studied by this system. Two distinct chiral separation mechanisms were proposed and good agreement was obtained between the results predicted by these mechanisms and the experimental data. Also, the use of this technique to determine solute-ligand equilibrium binding constant (K$\sb{\rm a}$) in free solution was examined. Several general guidelines in terms of sample size, ligand concentration range and applied voltage were developed for the application. Based on the equation derived for systems with 1 1 binding, good agreement was noted between the equilibrium constants measured in this work and those reported previously by other methods. In addition, two new terms (mobility ratio, M and migration time ratio, R$\sb{\rm t}$) were developed for obtaining a more reliable description of analyte migration in CE system. These terms can be use to cancel out the fluctuations due to the viscosity changes in CE running buffer. Section 3: Dicamba degradation study. The degradation of herbicide dicamba by bacteria Pseudomonas maltophilia DI-6 strain was studied by using CE, GC/MS and FAB/MS. A simple and fast CE method was developed for the analysis of dicamba degradation sample and confirmed that 3,6-dichlorosalicylic acid (DCSA) is one of its major degradation products. Also, the present studies clearly show that in addition to the previously established demethylation pathway through DCSA, there is another degradative pathway which has the direct dehalogenation of dicamba as its first step. And three types of bacteria enzyme activities (demethylation, methylation and dehalogenation) were observed in the degradation of dicamba by P. maltophilia DI-6.
Subject Area
Agricultural engineering|Chemical engineering|Biomedical research
Recommended Citation
Yang, Ju, "Theory and application of affinity liquid chromatography and capillary electrophoresis" (1995). ETD collection for University of Nebraska-Lincoln. AAI9600762.
https://digitalcommons.unl.edu/dissertations/AAI9600762