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 combination allows a 500-nm spectral window to be acquired with 2-nm resolution. The limits of detection are 5 x 10(-14) M or 80 molecules for sulforhodamine 101 and 1.5 x 10(-13) M or 220 molecules for fluorescein in a 50 micron i.d. capillary. An electropherogram of a mixture of amino acids derivatized with both Bodipy 503/512 C3 and Bodipy 576/589 C3 demonstrates that the analytes can be differentiated on the basis of both emission characteristics and migration times. With the use of organic modifiers and other complex separation media, the spectral background increases as discrete spectral features appear; the wavelength-resolved backgrounds of a variety of common CE separation conditions are presented.
Abstract. The ability of the cellular biochemist to understand events occurring in small subsections of an individual cell depends on available analytical techniques. Two unique capillary electrophoresis (CE) detection schemes for trace level peptide analysis are characterized: an on-line, multichannel, laser-induced fluorescence system and a postcolumn radionuclide system. The fluorescence system has picomolar concentration detection limits (and zeptomole mass limits of detection) for tagged amino acids and peptides. Preliminary results are presented for a method to enhance the ability to fluorescently tag low concentrations of amino acids and peptides from neuronal subsections. The procedure takes advantage of the high local concentration of peptides within a synaptic vesicle to enhance the derivatization reaction. The other detection method described involves a post-column 35S detector to examine methionine or cysteine containing peptides. This system deposits the effluent from the separation capillary onto a membrane treated with a solid scintillator. After drying the membrane, the photon emission is imaged with a solid-state integrating detector. The sensitivity for 35S labeled components is in the attomole range.
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