FLUORESCENT PROBE INVESTIGATIONS OF MICROENVIRONMENTS OF ANALYTICAL INTEREST (REVERSED-PHASE, POLYETHYLENIMINE, POLARIZATION)
Fluorescent probes were applied to investigate three systems. Reverse phase chromatographic surfaces were studied using ion pairs. Variables were cation reagent structure and concentration, bonded phase (methyl, octyl, octadecyl, and phenyl), and solvent (water or methanol). Emission wavelength shifts for the anionic polarity probe, ANS, (8-anilino-napthalene-1-sulfonate) reflect the nature and extent of lipophilic interactions. Tetramethylammonium promoted ANS penetration into surface structure. Tetrabutylammonium overcame aqueous surface alkyl aggregation, which greatly enhanced ANS-surface interaction of C18. For the other phases at high cation concentrations there was lipophilic interaction between ANS and cation. High concentrations of small cations excluded ANS from the surface, as did all levels of trimethylmyristylamine cation. Methanol solvation reduced lipophilic interactions with alkyl surfaces. Pi-pi interactions were important with the phenyl surface. Results are consistent with the ion interaction retention model for ion pairing chromatography, which is based on double layer formation on a dynamic surface. Polyelectrolyte-counterion binding strength and proximity were studied using a three component system: metals bound to polyethylenimine (PEI) and pyrenesulfonate counterion probes. Metals altered rates of excited state processes and defined binding environment. Variables were net charge on metal and probe and metal-amine complex properties. Cu(II)-PEI efficiently and selectively quenched probes. Ground state dimerization in Zn(II)-PEI implied territorial binding involving lipophilic interactions with PEI and between probes was important. Quenching and excimer formation in Ag(I)-PEI were due to more than net charge since protonated sites did not alter emission. Fluorescence polarization was used to detect intramolecular energy transfer between equivalent fluorophors in crown ethers, metal complexes, and simple organic molecules. Energy transfer randomizes the transition moment of emission relative to that of excitation, thereby decreasing polarization. In dilute glycerol solutions intermolecular depolarization is eliminated. A simple model based on Forster energy transfer theory was developed to distinguish molecules with different numbers of fluorophors and to obtain average angles between fluorophors, based on the extent of polarization differences.
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