Capillary electrophoresis with wavelength-resolved fluorescence for analysis of peptides
Timperman, Aaron Thomas
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Permalink
https://hdl.handle.net/2142/19433
Description
Title
Capillary electrophoresis with wavelength-resolved fluorescence for analysis of peptides
Author(s)
Timperman, Aaron Thomas
Issue Date
1995
Doctoral Committee Chair(s)
Sweedler, Jonathan V.
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Analytical
Language
eng
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.
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