Capillary Electrophoresis

Capillary Electrophoresis (CE) is an analytic separation technique used in different research areas such as biotechnology, pharmacy and medicine. This technique allows ion and charged molecule separation based on mobility differences in short periods of time with high efficiency and low solvent consumption. The name of the technique is due to the site where the separation takes place: a capillary, which is a tube with inner diameters ranging from 10 to 100 micrometers.

History

Capillary electrophoresis is a relatively new technique. The first system was developed in 1965 by Hjertén with the aim of separating proteins, nucleic acids and inorganic ions. However, the potential of the technique was further explored in 1980 by Jorgenson and Lukacs, who published high resolution separations with a simple homemade system.

Instrumentation

CE instrumentation is relatively simple. As can be seen in Figure 1, it consists of two platinum electrodes (anode and cathode) connected to a high voltage power supply and a fused silica capillary tube, whose ends are immersed into a reservoir containing buffer solution. For detection, the capillary tube must have an optical window, simply made by removing the polymer coating. This window is aligned with the detector, which is often a UV.

Figure 1. Schematic diagram of CE instrumentation

Sample Injection

In order to introduce the sample into the capillary, the inlet buffer reservoir is replaced by another containing the sample. After that, there are two possible methods of injection: electrokinetic, also known as electromigration, or hydrodynamic. The first one involves applying voltage and is usually used when the medium is viscous. The second is the most employed method and can be carried out in three ways:

• By applying pressure in the inlet
• By applying vacuum in the outlet
• By siphoning, which includes elevating the inlet reservoir in relation to the outlet

Modes of Operation and Applications

A buffer solution can be modified by adding substances, which leads to alternative mechanisms of retention and therefore to different modes of capillary electrophoresis. The modes or methods that can be mentioned are: Capillary Zone Electrophoresis (CZE), Micellar Electrokinetic Chromatography (MEKC), Capillary Gel Electrophoresis (CGE), Capillary Isoelectric Focusing (CIEF), and Capillary Isotachophoresis (CITP). If the capillary is filled with a stationary phase, as in HPLC, the method is called Capillary Electrochromatography (CEC). CE is used in multiple areas, and its applications depend on the mode:

• CZE has been employed in bioscience in order to separate peptides and proteins and also for the separation of organic acids and inorganic ions.
• MEKC is used for the separation of charged solutes as in CZE, but also neutral ones; some examples are amino acids, vitamins, pharmaceutical products, nucleotides and explosives.
• CGE is usually used in biological science for the separation of macromolecules such as proteins and nucleic acids.
• CIEF is the mode used to separate peptides and proteins based on their pI.
• CITP is useful for analyzing cations and anions simultaneously.

Bibliography

-Delfino, M. R. (2003). Electroforesis Capilar. Teoría, Técnica y Aplicaciones. Editorial universitaria de la Universidad Nacional del Nordeste.
-Heiger, D. (2000). High performance capillary electrophoresis, an introduction. Agilent technologies.