AN INVESTIGATION INTO THE ANALYTICAL UTILITY OF CHANGES IN POLARIZATION ACCOMPANYING ANALYTICAL DERIVATIZATION REACTIONS (FLUORESCENCE)

Derivatization reactions between amines, alcohols and carbonyl groups with various reagents were studied. The goal of the work discussed in this dissertation was to generate basic data to know whether and in what context changes in fluorescence polarization are useful for following analytical derivatization reactions. Reactions between primary amines and fluorescein isothiocyanate were initially studied. Changes in polarization were observed upon conjugation for many of the amines primarily due to changes in fluorescence lifetime. This reaction was also studied in varying amounts of glycerol to judge the effect of glycerol on the reaction rate. Optimum percentages of glycerol were determined. Rate data of reactions of fluorescein isothiocyanate with aniline and p-chloroaniline are reported. Reactions between amines and alcohols with dichlorotriazinyl fluorescein were studied next. Minimal changes in polarization were observed upon conjugation because the lifetimes were very similar to that of the reagent. Glycerol was a problem as it reacts rapidly with the reagent. In a set of kinetics experiments with polyethyleneimine, changes in polarization were observed but could not be distinguished from randomness brought about by the experimental conditions. Dansyl chloride was more difficult to work with than either of the fluorescein compounds due to its sensitivity to environment. This was illustrated by dramatic changes in lifetime as the amount of glycerol in the solution was varied for Perrin plots. The final study involved dansyl hydrazine and reactions with carbonyls. Polarizations again changed upon conjugation due to lifetime changes. Spectral shifts were also noted upon conjugation to two carbonyl compounds. Rate data for reactions with anisaldehyde are reported. A different TLC solvent system is suggested for separation of conjugate and free dansyl hydrazine