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.

PRINCIPLES

Capillary electrophoresis separates molecules due to their electrophoretic mobilities. A molecule's electrophoretic mobility depends on its charge and how much it is attracted or repelled by the voltage as well as the frictional drag force that resists movement. Friction is proportional to the radius of the molecule. Thus, electrophoretic mobility is based on size and charge. The velocity a charged molecule travels down a capillary is the product of its electrophoretic mobility and the applied electric field. Higher voltages therefore lead to faster velocities and faster separations.

Most capillary electrophoresis instruments are set up with the negative voltage at the detector end and the positive voltage at the inlet. This means that positively-charged molecules migrate towards the cathode at the end, while negatively-charged molecules migrate the other way. All molecules are seen at the detector however, because there is a bulk fluid flow called electroosmotic flow. The migration order is thus positively-charged, neutral, and then negatively-charged molecules.

Electroosmotic flow is caused by applying a high voltage to a small glass capillary filled with a salt solution. The positively-charged ions in the salt solution form a double layer with the negatively-charged silanol groups on the walls of the glass. When a negative voltage is applied to the end of the capillary, it pulls the cations from the double layer, which also pulls the solution around it due to frictional forces. This type of flow is plug-shaped and leads to less band-broadening than the parabolic-shaped flow plugs of HPLC.

Neutral molecules all flow at the same rate as the electroosmotic flow. However, a pseudo-stationary phase can be added to the run buffer to form micelles that molecules can partition in and out of. A typical pseudo-stationary phase is sodium dodecylsulfate. The micelles are negatively-charged on the outside, so they have an electrophoretic mobility, so the time spent in the micelle determines the migration time. This form of capillary electrophoresis is called micellar electrokinetic chromatography (MEKC).

Detection in CE is similar to that for HPLC. UV-Vis is general and does not require tagging as long as the molecule has a double bond. However, the absorbance depends on the path length, which is small for a 50-µm capillary. A bubble cell or z-cell will increase the path length. Laser-induced fluorescence is a more sensitive detection method. A laser is shone through a window in the capillary and fluorescence of the product measured. While fluorescence provides very high sensitivity, it generally requires molecules to be tagged because most are not fluorescent. Electrochemical detection and electrospray mass spectrometry detection are gaining in popularity. The issue with either of these detectors is that the high voltage from the separation must be brought to ground before the detection, as electrochemistry and electrospray require the application of a voltage and the CE voltage can interfere. New methods of decoupling the CE voltage, using electrodes to drain the current or a small crack in the capillary, are overcoming these challenges.

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:

1. By applying pressure in the inlet

2. By applying vacuum in the outlet

3.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:

1. CZE has been employed in bioscience in order to separate peptides and proteins and also for the separation of organic acids and inorganic ions.

2. 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.

3. CGE is usually used in biological science for the separation of macromolecules such as proteins and nucleic acids.

4. CIEF is the mode used to separate peptides and proteins based on their pI.

5. CITP is useful for analyzing cations and anions simultaneously.