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 increased optical path length. While extensively characterized for use in absorbance detection, they have not been adequately assessed for applications of laser-induced fluorescence (LIE) detection. Similar advantages (e.g. improved detectability) are expected with LIE, in addition to simplifying optical alignment. Thus, experiments were conducted to make comparisons between conventional and ELP capillary formats. In the first experiment, detection sensitivity was compared. The larger dimensions of the ELP capillary allowed efficient, far field spatial rejection of scattered light with an adjustable, precision slit. The greater optical path length permitted wider slit settings, thereby improving signal transmission by several fold. This experiment also revealed that 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 μm i. d. ELP capillaries, representing a significant improvement in ease of alignment over the conventional case. To further illustrate this feature, profiles of capillary wall scatter/fluorescence were generated by linear translation of the photomultiplier assembly in the far field while maintaining a constant slit width. These profiles clearly illustrate the improved spatial characteristics of the ELP capillary 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, A second pharmokinetic application of ELP capillary was also investigated. This preliminary study probed the applicability of the ELP capillary for increased sensitivity of the antipsychotic drug CP-88,059 and a related metabolite.