Dual wavelength excitation fluorescence detector for capillary electrophoresis using a pulsed bi-colour light emitting diode.

A simple and novel two-colour fluorescence detector for capillary electrophoresis was created using a single bi-colour light emitting diode (LED), multi-band pass excitation and emission filters and a single detector. Excitation light from a blue/red (470/635 nm) bi-colour LED was filtered through a 390/482/563/640 nm multi-band bandpass filter, with emitted light filtered through a 446/523/600/677 nm multi-band bandpass filter before being detected using a photon counting detector. Sequential pulsing of the blue/red LED and deconvolution of the collected fluorescence data allowed extracted electropherograms to be obtained corresponding to excitation with the blue and red LEDs. Optimisation of the pulsed LED conditions revealed an optimum LED on-time of 50 ms, off-time of 30 ms with a pulsed current of 40 mA, giving an effective data acquisition rate of 6.25 Hz. The characteristics of this system were validated by the simultaneous separation and determination of six fluorescent dyes: fluorescein, FITC, coumarin 334, dibromo(R)fluorescein (Ex/Em 470/525 nm), and Cy 5 and the Agilent Bioanalyser DNA dye (Ex/Em 635/670 nm). Under optimum conditions, the detection limits for FITC, fluorescein and Cy 5 were 69 nM, 42 nM and 289 nM (S/N = 3), respectively. These were lower than those obtained with continuous operation of the individual wavelengths at a constant current of 20 mA, but were slightly higher than those obtained using dedicated single wavelength filter combinations designed specifically for use with these fluorophores. The intraday repeatability (n = 6) of migration times was less than 1.0% and less than 3.4% for peak areas, while interday (n = 3) migration time and peak area reproducibility were less than 0.9% and 3.6%, respectively. This simple detector is capable of performing quantitative two-wavelength excitation without the need for complex optics and light source configurations.