Evaluation of extended light path capillaries for use in capillary electrophoresis with laser-induced fluorescence detection

An interesting development in improved capillary electrophoresis (CE) detection sensitivity is the Hewlett-Packard extended light path capillary (ELP). These capillaries have a ''bubble'' in the flow path at the detection zone which provides an increased optical path length. Although well characterized for use in absorbance detection, they have not been adequately assessed in the laser-induced fluorescence (LIF) detection mode. Similar advantages (e.g., improved detectability) are expected with LIF, in addition to simplifying the optical alignment. Therefore, experiments were conducted to make comparisons between conventional and ELP capillary formats. First, the detection sensitivity was compared with both formats by adjusting the background level (via fluorescence emission slit width adjustments) to a constant value. The larger dimensions of the ELP capillary allowed efficient rejection of the background with a wider slit setting, improving signal transmission several-fold. Second, alignment of the ELP capillary with the excitation beam and collection optics was significantly simplified. Clean transmission of the excitation beam was easily accomplished with both 50 and 25 mu m I.D. ELP capillaries, representing a significant improvement in ease of alignment over the conventional case. To illustrate this feature further, profiles of capillary wall scatter/fluorescence were generated by linear translation of the photomultiplier assembly at a constant slit width. These profiles clearly illustrate the improved spatial characteristics relative to conventional capillaries. The rapid, high-sensitivity separation of the metabolic enzyme markers 7-ethoxycoumarin and its metabolites in a liver slice incubate is shown as a practical application.