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Studies of novel tandem techniques using RPLC, adsorption chromatography and detection methods for analysis
Abstract
This dissertation represents the study and integration of column switching in the analysis of biological and environmental compounds. Areas of study in this work include: the determination of MHPG in human urine, the determination of atrazine and its degradation products in water, studies of binding site heterogeneity in split-peak chromatography and chromatographic immunoassays, and optimization of a chemiluminescent reaction for use in HPLC post-column detection. The studies with MHPG (3-methoxy-4-hydroxyphenylethylene glycol) resulted in a method for determination of total and free MHPG in urine using reversed-phase liquid chromatography (RPLC) with electrochemical detection. The chromatographic separation was made faster by the using of column switching to remove any late eluting peaks that would increase analysis time significantly. The determination of atrazine and its degradation products used of column switching to couple, high performance immunoaffinity chromatography (HPIAC) and RPLC. The results of the HPIAC/RPLC method agreed well with the results of a reference GC/MS method. In addition, the HPIAC/RPLC was shown to be able to quantitate these compounds at the parts-per-trillion range in water. The theoretical studies of heterogeneity showed that binding site variations can affect split-peak adsorption behavior. Simulations suggested a linear equation describing such behavior. Simulations and experiments showed that the presence of a slow binding site decreases the amount of solute adsorbed to the column under all conditions. For a sequential addition immunoassay (SAIA), computer simulations predicted that heterogeneity results in a larger dynamic range for sample quantitation. However, heterogeneity generally resulted in small changes in the assay results versus those obtained in homogeneous assays. Thus heterogeneity in the stationary phase, should not be a great hindrance in the use and performance of these assays. The last topic, the optimization of the chemiluminescence reaction of acridinium esters for post-column determinations was performed for the detection of labeled compounds can be determined at sub-attomole levels. The optimal levels determined for phenyl acridinium esters were 1 M sodium hydroxide, 0.02% (w/v) hydrogen peroxide and 1% (w/v) Triton X-100. In addition, the post-column system gave optimal behavior if a total flow rate greater than 3.0 mL/min was used for the post-column reaction mixture.
Subject Area
Analytical chemistry|Biochemistry
Recommended Citation
Rollag, John Gregory, "Studies of novel tandem techniques using RPLC, adsorption chromatography and detection methods for analysis" (1996). ETD collection for University of Nebraska-Lincoln. AAI9715981.
https://digitalcommons.unl.edu/dissertations/AAI9715981