Capillary Electrophoresis

What is the capillary electrophoresis technique?

Capillary Electrophoresis (CE) is a method commonly used to separate materials based on their electrophoretic mobility. In this process, the movement of ions, induced by an electric current, aids in the separation of components. Electrophoretic mobility depends on molecular charge, viscosity and atomic radius. The velocity of particle movement is directly proportionate to the electric field applied, thus a stronger field results in swifter movement. On the other hand, neutral species remain unaffected, while only ions possessing electric charge pass through the field.

When two ions are of equal size, the ion with a greater charge will exhibit faster movement. In the case of ions carrying the same charge, the smaller particle experiences less friction and typically moves more expeditiously. CE has gained significant popularity in recent times due to its remarkable efficiency, selectivity, resolution and sensitivity. Consequently, it has emerged as a widely employed method for analyzing complex mixtures of molecules, leading to numerous advancements and substantial improvements in the affected context.

Types of capillary electrophoresis technique

The capillary electrophoresis technique encompasses several distinct types, which will be presented in the subsequent sections.

Capillary zone electrophoresis (CZE)

In this type of capillary electrophoresis, only one capillary and a buffer without any anticonvective environment are needed for separation. The analytes are divided into bands, the velocity of which depends on both electrophoretic flow and electroosmotic flow. The electroosmotic flow moves towards the cathode and upon polarity reversal, analytes possessing superior electroosmotic mobility to the electroosmotic force traverse the outlet. Coated capillaries serve to augment the separation capacity of materials that adhere to the fused silica surfaces.

Capillary gel electrophoresis (CGE)

In the capillary gel electrophoresis (CGE) method, the separation is conducted within a gel-filled capillary, akin to gel electrophoresis. In this technique, molecules are segregated based on their molecular size, with smaller molecules moving more freely within the gel and thus migrating more expeditiously than larger molecules. In general, capillary gel electrophoresis finds utility in the separation of proteins, DNA fragments and other biological macromolecules possessing akin charge-to-mass ratios, predicated on their molecular size.

Micellar electrokinetic capillary chromatography (MEKC)

The MEKC technique involves the separation of solutes in an electrolyte solution that contains a surfactant at a concentration higher than the critical micelle concentration (CMC). This technique combines electrophoresis and chromatography and is applicable to both neutral and charged solutes. The solute molecules are distributed between the pseudo-stationary phase, which consists of micelles, and the aqueous buffer based on the partition coefficient of the solutes. Commonly used surfactants in MEKC include sodium dodecyl sulfate, cetyltrimethyl and ammonium salts. In neutral and alkaline pH conditions, there is a strong electroosmotic flow and the ions of the separation buffer move towards the cathode. When sodium dodecyl sulfate (SDS) is used as a surfactant, the anionic micelle migrates towards the anode, thereby reducing the overall migration rate of the micelle in the volume flow of the electrolyte solution.

Capillary electrochromatography (CEC)

CEC represents a fusion of separation methods that employs the fundamental principles of capillary electrophoresis and chromatography, specifically high performance liquid chromatography (HPLC). Within this methodology, the analytes are segregated based on disparities in partition ratio between the mobile and stationary phases or as a result of electrophoretic mobility. Unlike pressure-driven mechanisms, the mobile phase traverses the chromatographic bed via electroosmotic force. CEC boasts a multitude of advantages in comparison to HPLC and capillary electrophoresis. Noteworthy applications encompass enantiomeric separation as well as the segregation of amino acids, proteins, peptides, and carbohydrates. Broadly speaking, CEC predominantly finds utility in the pharmaceutical sector for identifying acidic and basic drugs, as well as in the industrial domain which entails polymer analysis.

Capillary isoelectric focusing (CIEF)

In this type of capillary electrophoresis methodology, the motion of charged molecules transpires as a consequence of the existence of an electrical field in isoelectric focusing, which arises from a pH gradient that is brought about by ampholytes possessing a pI value (i.e., the isoelectric point) encompassing a broad spectrum and is dissolved within the separation buffer.

Capillary isotachophoresis (CITP)

In a general, the CITP technique was devised with the objective of segregating serum lipoproteins. This technique is typically employed within clinical laboratories for the determination of lipoprotein levels found in human serum.

Instruments used in the capillary electrophoresis technique

In the capillary electrophoresis technique, a variety of instruments are employed, which we shall introduce in the subsequent section.

Power Supply

In the CE technique, an energy source is requisite for the generation of a controlled, direct electric current with a high voltage.

Buffer reservoirs

In capillary electrophoresis two tanks for buffering are necessitated, with the requirement that they be maintained at an equivalent height. These reservoirs contain distinct solutions for the anodic and cathodic components.

Electrodes

In the capillary electrophoresis technique, two electrodes (cathode and anode) submersed in the buffer tanks and connected to the power source are utilized for the establishment of an electric current

Capillary

The capillary employed in the CE technique is composed of fused silica and typically has a diameter of less than 100 microns.

Viewing window

In this technique, the presence of an optical viewing window that is appropriately aligned with the detector is necessary.

Injection system

The injection system is a vital component of the device that is specifically engineered for the purpose of introducing sample and buffer into the capillary. In certain instances, the injection method can be automated to enhance precision of operation. Common techniques employed for injecting samples encompass gravity, pressure or vacuum, as well as electrokinetic methods.

Detector

In the CE technique, a detector is employed to assess the amount of substance that traverses through the capillary during a specific time interval, Conductivity detection, fluorimetry, absorption spectrophotometry (UV and visible), amperometric or mass spectrometric detection.

Thermostatic system

The role of the thermodynamic system in CE is to ensure a consistent temperature within the capillary.

Recorder

This segment of the electrophoresis capillary is designed for the purpose of recording data.

Capillary electrophoresis technique advantages

Highly efficient
Less time-consuming
this technique finds application in a wide range of areas, particularly in the detection of macromolecules like DNA, RNA and proteins.
Ability to perform the reactions automatically

Capillary electrophoresis technique disadvantages

Despite the numerous advantages presented by the capillary electrophoresis technique, this approach also encompasses certain drawbacks, which include:

Complexity in altering capillaries: Given the dimensions of the capillaries, the process of removing or substituting them can prove to be challenging under certain circumstances. Thus, any modifications to the capillaries must be executed by an expert.
Heightened sensitivity of the capillary electrophoresis technique: Minute alterations such as the dimensions of the capillaries and the selection of analytes can exert significant influence on the outcomes of the capillary electrophoresis technique. Consequently, CE exhibits a greater degree of sensitivity compared to alternative methodologies.