| Capillary electrophoresis (CE) is a widely accepted separation method for biological applications due to its small sample compatibility. Small sample volumes demand high-sensitivity detection, most commonly laser-induced fluorescence (LIF). Typical lasers used in LIF are large and expensive; qualities which are contrary to the nature of CE. This thesis investigates alternative light sources for high-sensitivity fluorescence detection.; The first light source explored was the violet diode laser, emitting 5 mW at 400 nm. Compared to gas-phase lasers typically used for LIF, the violet diode laser is considerably smaller, about half the price, but significantly less powerful. To demonstrate the abilities of the violet diode laser, labelling and detection of the protein myoglobin was performed. The non-fluorescent heme group of myoglobin was replaced with a fluorescent porphyrin. The labelling is unaffected by a 2000-fold excess of bovine serum albumin, a potential interferent, and produces a detection limit of 50 nM for myoglobin.; Another light source with reduced cost and size is a light-emitting diode (LED). At {dollar}1 to {dollar}25, the cost of an LED is trivial and its size is approximately an order of magnitude smaller than most lasers. The optical properties of LEDs present several challenges due to their large emitter size and highly divergent, polychromatic light output. An epi-fluorescence detection geometry focussed the LED light to a ∼ 150 mum spot, slightly larger than the capillary inner channel. The LEDs' polychromatic light gives an extremely high background signal due to scatter of the LED light at the detection wavelength. A bandpass filter between the LED and the capillary reduces this background signal and increases the signal-to-noise of the detector by a factor of ≥ 70. A detection limit of 3 nM was achieved using {dollar}1, low-power LEDs and 3 pM with a {dollar}25, high-power LEDs. This level of sensitivity rivals that achieved using gas-phase lasers. The detector was applied to monitoring protein purification as well as DNA separations. DNA was detectable at 40 mug/mL. In sacrificing perhaps an order of magnitude in detection limit, LEDs give two or three orders of magnitude savings in cost, relative to gas-phase lasers. |
