Capillary Electrophoresis: Overview

Capillary electrophoresis (CE) is an analytical liquid-base separation technique. It separates biomolecules or ions in a capillary channel based on their electrophoretic mobility under the influence of an electric field.

The movement of the molecule during the application of high voltages is based on the electrophoretic mobility and the buffer’s electroosmotic flow (EOF) mobility inside the capillary. The analyte’s or solute’s electrophoretic mobility depends on its molecular size and shape and electric charge.

The capillary electrophoresis system is widely used in analytical biochemical processes, such as nucleic acid analysis, coenzyme separation, protein identification, genome sequencing, and DNA fingerprinting.

CE instruments used to perform the assays include:

• Fused silica capillary: These capillaries contain an ionizable silanol group. At pH 2.0, they create a double layer of the negatively charged capillary wall for cation accumulation. During voltage supply, the positively charged ions migrate toward the cathode, facilitating molecule separation.
• Electrodes: The system has two electrodes, a cathode (that attracts cations) and an anode (that attracts anions) immersed in a buffer reservoir, which is connected to the power supply.
• Buffer reservoir: It contains two containers, connected to a power source, for two buffer solutions (cathodic and anodic buffer solutions).
• Detector: Most commonly used detector in the CE system is an ultraviolet-visible absorption type. The high-resolution data obtained from it is displayed on an output device, such as a computer as an electropherogram (separated molecules appear as peaks with different migration times).

Different detection methods are used in different assays. For example, fluorescent detection is used in DNA sequencing, which has high sensitivity and improved selectivity. However, UV or UV-Vis absorbance systems remain most commonly used in workflows.

• Injection system: Used to add buffer or sample to the capillary. The most common methods used to perform the process are electrokinetic, vacuum, or gravity.
• Thermostatic system: Maintains a constant temperature inside the capillary during the workflow.

Figure: Schematic diagram of the instruments involved in Capillary electrophoresis workflow.

In this article, we will cover how capillary electrophoresis works, its types, and its applications in different life sciences industries.

How Does Capillary Electrophoresis Work?

The capillary of the CE system is made of fused silica, having inner negatively charged layers. Thus, when high voltage is supplied at the end of the capillary tube, the molecules in a mixture start separating based on their ionic mobility and interaction with the liquid phase or electrolyte medium.

The molecules or ions with smaller sizes or higher charges move faster in the molecules than those larger in size or having more charges. Additionally, the molecules can also be concentrated by creating a gradient in the pH or conductivity of the electrolyte solution.

Figure: An illustration of an electroosmotic flow of ions between a double layer of ionized SiOH reagent in the capillary.

The separated molecules are then detected by a detector, which displays them on screen as distinct peaks as a function of migration time. The electrophoretic mobility of molecules in response to an electric field depends on their charge, radius, and solvent viscosity.

Capillary electrophoresis can also be combined with mass spectrometry (as first done by Richard D. Smith) to analyze small molecules with high sensitivity. The technique is widely used because of its high-resolution separation approach and efficiency when compared to other separation techniques like HPLC (High-performance liquid chromatography).

Types of Capillary Electrophoresis

Here’re five types of commonly known capillary electrophoresis:

• Capillary zone electrophoresis (CZE): One of the most commonly used capillary electrophoresis techniques. It’s faster and easier than the other techniques. It only requires a buffer and no anti-convective medium. The polymer-coated capillary helps in the separation of molecules in a mixture.
• Capillary isoelectric focusing (CIEF): Charged molecules move because of an electric field, within a pH gradient created by using different ampholytes with isoelectric point values that dissolve in a separation buffer.
• Micellar electrokinetic chromatography (MEKC): A combination of electrophoresis and chromatography technique. It separates both neutral and charged molecules. The electrolytic solution used in the technique contains a surfactant (such as sodium dodecyl sulfate or SDS) in a concentration higher than the critical micellar concentration (CMC). The molecules or ions are distributed between the aqueous buffer and the pseudo-stationary phase of the micelle based on their partition coefficient.
• Capillary gel electrophoresis (CGE): Involves the use of a capillary filled with a gel that acts as a sieve for the separation of molecules based on their size. It’s similar to gel electrophoreses and used t separate biomolecules, such as proteins and DNA fragments.
• Capillary electrochromatography (CEC): Combines capillary electrophoresis and HPLC and the molecules are separated based on the partition ratio between two mobile and stationary phases of the system. It’s mainly used in the pharma industry to distinguish acidic and basic drugs.

What is Capillary Electrophoresis Used For?

Capillary electrophoresis (CE) is a powerful technique used in molecular biology and pharma labs to efficiently separate and detect molecules, such as oligonucleotides, nucleic acids, peptides, and steroid hormones.

DNA Analysis

Capillary electrophoresis is a high-throughput technique used for DNA analysis to achieve faster high-resolution data with small sample volumes. The two most common CE modes used for the process include Free-zone and gel-facilitated sieving. Additionally, CE systems can also be used to achieve outstanding separation efficiencies of biomolecules including peptides, amino acids, and amines.

Affinity Testing

Capillary electrophoresis is an efficient method to analyze the non-covalent interactions between ligands and analytes on the basis of their electrophoretic mobility changes in the medium. The technique has been widely applied to studying different biomolecules, including polysaccharides, proteins, and hormones.

What Industries Use Capillary Electrophoresis?

For its efficient separation, improved selectivity, easy coupling with other techniques, and data reproducibilities, such as a mass spectrometer or Raman spectroscopy, capillary electrophoresis is widely used in many life sciences labs.

Pharmaceutical

Capillary electrophoresis is used during the preparation and analysis of standards solution of small molecules in drugs. CITP is crucial in detecting cholesterol levels in clinical laboratories for lipid profile analysis. Additionally, capillary electrophoresis is used to perform the analysis of vitamins and minerals present in human serum.

Food Industry

Capillary electrophoresis is utilized for examining various food types, such as fermented food and beverages, to identify pigments, flavonoids, carbohydrates, vitamins, proteins, and color. Additionally, CE can be used for detecting contamination of bacteria and viruses by examining their nucleic acid (DNA and RNA) in a given sample.

Forensic Science

Capillary electrophoresis (CE) is employed in the field of forensic science to amplify and detect DNA fragments through the polymerase chain reaction (PCR). Further, it’s also used to separate and detect short tandem repeat (STR) alleles and examine mRNA to determine biological fluid or the source of tissues in forensic specimens.

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Capillary electrophoresis is a powerful and efficient method to separate biomolecules, such as peptides, proteins, and nucleic acids, based on their size and charge. The molecules are separated between two electrodes in the presence of a strong electric field.

Some commonly known CE variants include Capillary isoelectric focusing (CIEF), Capillary zone electrophoresis (CZE), and Capillary gel electrophoresis (CGE).

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