Colinear laser-induced fluorescence detector for capillary electrophoresis. Analysis of glutamic acid in brain...

Experiments with capillary electrophoresis using a laser-induced fluorescence detector with a colinear optical arrangement demonstrated several important points. First, increasing the numerical aperture of the microscope objective that is used simultaneously for focusing the excitation laser light as well as collection of emitted fluorescence enhances the signal used for the measurement of the emitted fluorescence and at the same time decreases the noise of interfering light. Second, detection of fluorescein-labelled amphetamine was performed at high-picomolar (10(-10) M) levels. Third, the signal-to-noise ratio of 280 found at the above-mentioned picomolar concentrations indicates that the measurement of low-picomolar concentrations (10(-12) M) of this compound in biological samples should be possible. Fourth, narrow-bore capillaries (5-10 microm internal diameter) were used to detect the neurotransmitters glutamic acid and aspartic acid as their naphthalene-2,3-dicarboxaldehyde derivatives in brain dialysates obtained from a freely moving rat. A mathematical model was developed to explain the relationship between numerical aperture, working distance, magnification of the lens, noise due to laser scattering and signal due to fluorescence. The model correctly predicted the observed values of photomultiplier tube current due to both laser scattering and fluorescence. The potential of the application of capillary electrophoresis with laser-induced fluorescence detection in the neurosciences is discussed.