Source: Laboratory of Dr. B. Jill Venton - University of Virginia

Capillary electrophoresis (CE) is a separation technique that separates molecules in an electric field according to size and charge. CE is performed in a small glass tube called a capillary that is filled with an electrolyte solution. Analytes are separated due to differences in electrophoretic mobility, which varies with charge, solvent viscosity, and size. Traditional electrophoresis in gels is limited in the amount of voltage that can be applied because Joule heating effects will ruin the gel and the separation. Capillaries have a large surface area-to-volume ratio and thus dissipate heat better. Therefore, the voltages applied for a capillary electrophoresis experiment are quite large, often 10,000–20,000 V.

Capillary electrophoresis is useful for high-performance separations. Compared to liquid chromatography, CE separations are often faster and more efficient. However, capillary electrophoresis works best to separate charged molecules, which is not a limitation of liquid chromatography. CE has a greater peak capacity than high-performance liquid chromatography (HPLC), meaning the separations are more efficient and more peaks can be detected. The instrumentation can be very simple. However, HPLC is more versatile and many stationary and mobile phases have been developed for different types of molecules.

1. CE Instrumentation Setup

1. Turn on the CE instrument and computer. Using the computer software, turn on the light source for UV analysis to allow it to warm up. Some software has an indicator when the lamp is ready for use (lamp icon turns color).
2. Make a methods file. Set the important parameters for running the CE. In this analysis the temperatures of the cartridge and sample storage are 35 °C. The wavelength for UV detection is 214 nm.
3. Write a time program. The program general

Electropherograms collected for diet Pepsi and Pepsi samples are shown in Figures 1 and 2, respectively. The three peaks for caffeine, aspartame, and benzoic acid are observed in diet Pepsi and have similar migration times as the standards. For regular Pepsi, the caffeine peak is present but not the aspartame and benzoic acid peaks. The CE analysis is fast as the migration times are only 3–4 min.

The calibration curve for caffeine is shown in Fig

Capillary electrophoresis is used for many specialty separations. For example, it is used in the pharmaceutical industry for quality testing, to make sure there are no side products or interferents. CE is particularly useful for separating drugs with a basic amino group, as the walls of the capillary can be made neutral with an acidic pH and thus the drug will not stick to the capillary.