An axial approach to detection in capillary electrophoresis
The purpose of this research has been to develop and demonstrate visualization schemes which further the capabilities of capillary electrophoresis instrumentation. Our approach involves on-axis illumination of the compounds inside the capillary detection region and is applied to absorbance and fluorescence detection;Absorbance measurements were accomplished by focussing an incident beam of laser light into one end of the separation capillary. By utilizing signals collected over the entire length of the analyte band, this technique enhances the analytical path length of conventional absorbance detection sixty fold. The demonstrated instrument offers a fifteen-fold improvement in concentration limits of detection;Three fluorescence detection experiments are discussed; all of which involve the insertion of a small optical fiber into the capillary to introduce the excitation beam from a laser source. The first of these employs a high refractive index liquid phase to satisfy total internal reflectance along the capillary axis, thus greatly reducing light scatter from capillary walls. The second utilizes a charge-coupled device (CCD) camera for simultaneous imaging of an array of capillaries (a technique that may prove useful in high information throughput tasks such as genome sequencing). The third is basic study of fluid motion inside the capillary under conditions of pressure-driven and electroosmotic flow. In this study, the CCD is used to track nanometer-sized particles as well as fluorescent bands to give a quantitative, empirical assessment of the factors influencing capillary electrophoresis separations.