Principles of Capillary Electrophoresis

                         Capillary electrophoresis (CE) separations are carried out inside a capillary tube, which usually has a diameter of 50 μm to facilitate temperature control. The length of the capillary differs in different applications, but it is typically in the region of 20–50 cm. The capillaries most widely used are fused silica covered with an external protective coating. A small portion of this coating is removed to form a window for detection purposes. 

                     The ends of the capillary are dipped into reservoirs filled with the electrolyte. Electrodes made of an inert material such as platinum are also inserted into the electrolyte reservoirs to complete the electrical circuit. The capillary is filled with running buffer, one end is dipped into the sample, and an electric field (electrokinetic injection) or pressure is applied to introduce the sample inside the capillary. Migration through the capillary is driven by application of a high-voltage current (10–30 kV). The molecules are detected as they pass through the window at the opposite end of the capillary. The most frequently used detector is laser-induced fluorescence (LIF), which detects fluorochromes attached to the DNA molecules; alternative detectors include ultraviolet (UV) absorbance and fluorescence. 

                         Given the short path, detection requires monitoring by sensitive equipment such as chargecoupled device (CCD) cameras. The detectors are interfaced with computers responsible not only for collecting and displaying the data but also for maintaining the timing between filter wheels and controling timing exposures, readouts of the CCD cameras, and run-time processing of the CCD images and spectral data. The molecules are detected as fluorescent peaks as they pass through the detector. 

                       An electropherogram, which is a plot of the detector response with time, is generated. Because the area of each peak is proportional to the concentration of the DNA molecule, integrated peak areas are routinely used for semiquantification due to their greater dynamic range than peak heights.