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Title:Capillary electrophoresis with wavelength-resolved fluorescence for analysis of peptides
Author(s):Timperman, Aaron Thomas
Doctoral Committee Chair(s):Sweedler, Jonathan V.
Department / Program:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Chemistry, Analytical
Abstract:Wavelength-resolved fluorescence detection in capillary electrophoresis yields a powerful analytical tool for analysis of complex micro-environments. The design, development, and applications of wavelength-resolved fluorescence detection for capillary electrophoresis are reviewed.
A multichannel laser-induced fluorescence detector for capillary electrophoresis is described. The detection system combines yoctomole limits of detection with the simultaneous acquisition of entire fluorescence emission spectra. An Ar/Kr mixed gas ion laser provides great flexibility in excitation wavelengths and a holographic grating and charge-coupled device detector (CCD) combination allows a 500-nm spectral window to be acquired with 2-nm resolution. The limits of detection (LODs) are $5\times10\sp{-14}$ M or 80 molecules for sulforhodamine 101, and $1.5\times10\sp{-13}$ M Or 220 molecules for fluorescein in a 50 $\mu$m I.D. capillary. An electropherogram of a mixture of amino acids derivatized with both Bodipy 503/512 C$\sb3$ and Bodipy 576/589 C$\sb3$ demonstrates that the analytes can be differentiated on the basis of both emission characteristics and migration times.
A native fluorescence detection system for capillary electrophoresis is described that achieves low attomole detection limits and simultaneous acquisition of complete fluorescence emission spectra. The detection system employs a frequency doubled krypton laser operating at 284 nm for excitation, a sheath flow cell, a reflective f/1.2 microscope objective, an imaging spectrograph, and a CCD detector. The detection capabilities are characterized with tryptophan and tyrosine which have limits of detection (3s) of $2\times10\sp{-10}$ M and, $2\times10\sp{-8}$ M, respectively. Acquisition of the fluorescence emission spectrum provides the ability to distinguish three classes of peptides: those that contain tryptophan, tyrosine, or both tryptophan and tyrosine.
The combination of wavelength-resolved fluorescence detection and a fluorescent pH indicator, incorporated into the running buffer, is used as a novel on-column pH monitor. When the outlet vial is removed, rapid pH shifts of more than 3 units are observed for running buffers in the 4-8 pH range. A front of reverse migrating OH$\sp{-}$, and zones of stacking and depletion of the SNARF, pH indicator, are observed.
Issue Date:1995
Rights Information:Copyright 1995 Timperman, Aaron Thomas
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9624517
OCLC Identifier:(UMI)AAI9624517

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