How Does Capillary Electrophoresis Work

Capillary electrophoresis (CE) is an analytical separation method that uses an electric field to separate the components of a mixture. Basically, it is electrophoresis in a capillary, a narrow tube. Hence, the components of the mixture are separated based on their electrophoretic mobility. The three factors that determine the electrophoretic mobility of a particular molecule are the charge of the molecule, viscosity of the separation medium, and the radius of the molecule. Only the ions are affected by the electric field while the neutral species remain unaffected. The rate of a molecule that moves through the capillary depends on the strength of the electric field.

Key Areas Covered

1. What is Capillary Electrophoresis
     – Definition, Instrumentation, Methods
2. How Does Capillary Electrophoresis Work
     – Theory of Capillary Electrophoresis

Key Terms: Capillary Electrophoresis (CE), Capillary Electrophoretic Separation Methods, Capillary Tube, Charge, Electroosmotic Flow Electrophoretic Mobility

What is Capillary Electrophoresis

Capillary Electrophoresis refers to an analytical separation method by which the components of a mixture are separated based on their electrophoretic mobility. In early experiments, a glass U tube filled with gels or solutions were used. Capillaries were used after the 1960s.

Instrumentation

The capillary is made up of fused silica, having an inner diameter of 20-100 µm. A high voltage electric field is supplied to the ends of the capillary tube. The electrodes are connected to the ends of the capillary tube through an electrolyte solution or aqueous buffer. The capillary is filled with a conductive fluid at a certain pH. In addition to detectors and other output devices, some instruments are used for the temperature control of the system, ensuring reproducible results. The sample is introduced to the capillary by injection. The instrumentation of capillary electrophoretic system is shown in figure 1.

Figure 1: Capillary Electrophoresis – Instrumentation

Methods of Capillary Electrophoretic Separation 

Six types of capillary electrophoretic separation methods can be identified.

1. Capillary zone electrophoresis (CZE) – A free solution is used as the conductive fluid.
2. Capillary gel electrophoresis (CGE) – A gel is used as the conductive fluid.
3. Micellar electrokinetic capillary chromatography (MEKC) – The components of a mixture are separated by partitioning between micelles and the solvent/conductive fluid.
4. Capillary electrochromatography (CEC) – A packed column is used in exception to the conductive fluid. A mobile liquid is passed over the column along with the mixture to be separated.
5. Capillary isoelectric focusing (CIEF) – Mainly used to separate zwitterionic components such as peptides and proteins that contain both positive and negative charges. A conductive fluid with a pH gradient is used to separate the protein solution. Each protein migrates to the area with its isoelectric point within the pH gradient. At the isoelectric point, the net charge of proteins become zero.
6. Capillary isotachophoresis (CITP) – It is a discontinuous system. Each component migrates in consecutive zones, and the amount of the component is obtained by measuring the length of migration.

How Does Capillary Electrophoresis Work

Generally, the charged species begin to move in electric fields. The charge, viscosity, and molecular radius are the three factors that determine the electrophoretic mobility of a molecule in an electric field.

1. Charge – Cations (positively-charged molecules) move towards the cathode (negative electrode) while anions (negatively-charged molecules) move towards the anode (positive electrode).
2. Viscosity – The viscosity of the medium is opposite to the movement of molecules, and it is constant for a particular separation medium.
3. Radius of ion/molecule – Electrophoretic mobility decreases with increasing radius of the molecule.

Hence, if two molecules with the same size are subjected to electrophoresis, the molecule with the greater charge will move faster. The rate of migration of the charged species is increased with the increasing strength of the electric field. The mechanism of capillary electrophoresis is shown in figure 2.

Figure 2: Capillary Electrophoresis

Electroosmotic Flow (EOF)

The electroosmotic flow generates the mobile phase of capillary electrophoresis. In most cases, the capillary material is silica. Silica is hydrolyzed, yielding negatively charged SiO^– ions when the solutions with pH greater than 3 are passed through the capillary tube. Then, the capillary wall bears negatively charged layer. Cations of the solution are attracted to these negative charges, forming a double layer of cations on the negative charges. The inner cation layer is stable while the outer cation layer moves toward the cathode as a bulk flow of charged molecules. The bulk flow of cations occurs near the capillary wall during capillary electrophoresis. Electroosmotic flow near the capillary wall is shown in figure 3.

Figure 3: Electroosmotic Flow

The small diameter of the capillary wall contributes to maximizing the effect of EOF, aiding it to play a vital role in the movement of charged species in capillary electrophoresis. 

Conclusion

Capillary electrophoresis is an analytical separation method in which the charged species are separated based on their electrophoretic mobility. Generally, the size and the charge of the molecules serve as factors for the separation.

Reference:

1. “Capillary Electrophoresis.” Chemistry LibreTexts, Libretexts, 28 Nov. 2017, Available here.

Image Courtesy:

1. “Capillaryelectrophoresis” By Apblum –  (CC BY-SA 3.0) via Commons Wikimedia
2. “Capillary electrophoresis” by Andreas Dahlin (CC BY 2.0) via Flickr
3. “Capillarywall” By Apblum – english wikipedia (CC BY-SA 3.0) via Commons Wikimedia