LASER-INDUCED FLUORESCENCE AND FLUORESCENCE MICROSCOPY FOR CAPILLARY ELECTROPHORESIS ZONE DETECTION

A procedure to improve on-column fluorescence detection for capillary zone electrophoresis is reported. A fluorescence detector was built using an epillumination fluorescence microscope and an argon-ion air-cooled laser. The 488-nm line was isolated with a band pass filter to eliminate the ultraviolet line. A dichroic mirror which reflected wavelengths under 510 nm and a 0.75 numerical aperture (NA), 0.3 mm working distance objective of the microscope condensed the laser beam on a fused-silica capillary and a cross-shaped fluorescence spot was observed. The emitted light was collected with the same objective, filtered with a 520-nm high pass filter, a spatial filter and a notch filter, and focused on a photodetector. The photodetector was either a gallium-arsenide or a multialkali photomultiplier tube. The signal generated was fed to a current-to-voltage converter and registered on a strip chart recorder. The detector was tested with fluorescein-derivatized amino acids. For the fluorescein thiocarbamyl (FTC)-amino acids the limit of concentration detection varied according to the amino acid. Taking FTC-arginine as the best example, the limit of concentration detection (LOCD) was 3.75.10(-12) M. Assuming that 1 nl was injected, the limit of mass detection (LOMD) was 3.75.10(-3) amol or about 2250 molecules. This represents an improvement of about three orders of magnitude with respect to previous laser induced fluorescence on-column detection. The potential and advantages when using an epillumination fluorescence microscope with laser induced fluorescence as a detection system for capillary electrophoresis are discussed.