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Capillary electrophoresis instrumentation typically consists of several key components. A high-voltage power supply generates the electric field necessary for the separation by connecting to an anode (the positively charged electrode) and a cathode (the negatively charged electrode) located in buffer reservoirs at each end of the capillary tube. The system includes a sample vial, a fused silica capillary tube coated with polyimide for mechanical strength through which the sample components migrate during separation, and a detector to analyze the separated components.
The sample must be introduced into the capillary tube to initiate the process. This is achieved by detaching one end of the capillary and its electrode from the associated buffer reservoirs and placing them in the sample vial. Sample introduction can be accomplished through hydrodynamic injection, which involves applying pressure to the sample vial, or electrokinetic injection, which relies on an electric field to drive the sample into the capillary.
When current flows through the capillary containing a conductive buffer solution, it leads to Joule heating due to the narrow bore of the capillary column and the relative thickness of the capillary's walls. Joule heating refers to the heat generated as an electric current passes through a conductive medium—in this case, the buffer solution inside the capillary. It is directly related to the energy dissipation in the form of heat, as defined by the equation Q = I²Rt, where Q is the heat energy (in joules), I is the current, R is the resistance, and t is time. In Capillary electrophoresis, this heating can change the viscosity of the buffer solution, causing solutes in the center of the capillary to migrate faster than those near the walls, resulting in band broadening and degraded separation. Capillaries with smaller inner diameters generate less Joule heating, while those with larger outer diameters are more effective at dissipating heat.
A stacking technique may be employed to enhance detection sensitivity in cases where the sample concentration is low. This method involves injecting the sample into a solution with a lower ionic strength than the buffering solution, causing the sample components to concentrate at the interface between the two solutions.
Once the sample has been introduced, a high voltage is applied across the buffer system, prompting charged species to migrate toward the cathode through electroosmotic flow. The separation of components occurs based on their electrophoretic mobility within the electric field.
Various detectors, such as absorption, fluorescence, conductivity, and mass spectrometry, are commonly employed in capillary electrophoresis to detect and analyze the separated components.
