How Capillary Electrophoresis Works & How We Save You Time & Money

Though the specific components involved may vary slightly from model to model, there are several that are commonly found in CE instruments:

• Sample vial to hold the sample.
• Source and destination vials located on either ends of the capillary tube.
• Capillary tubing used to separate the molecules within the sample.
• A power supply and various electrodes to supply the charge needed for migrating the sample’s contents from end to end.
• A detector to observe the separation due to electrophoretic mobility. The mode of detection can be fluorescence-based or UV/VIS-based. However, CE systems can also be directly coupled with mass spectrometers or Surface Enhanced Raman Spectroscopy (SERS).
• A computer system to analyze and store the collected data.

Additionally, multiple methods of CE have been developed by altering the capillary buffer solution. Several of these methods are:

This is a fundamental part of capillary electrophoresis. In CE, there are columns within the capillary composed of silica, referred to as silica capillaries, that create a negatively charged inner capillary surface. They can be coated or uncoated. But, when they are coated, they’re typically coated with a polymer that can slow down the rate of flow within the capillaries. \n\nCations present in an ionic solution will migrate toward the negatively charged wall, forming an electric double layer. The generation of energy across the column causes cations to migrate towards the cathode and away from the anode. \n\nElectroosmotic flow occurs as the cations gather at the capillary walls, dragging the bulk of the sample towards the cathode. This flow is necessary because it causes movement of nearly all ions present, regardless of charge, in the same direction. It allows for simultaneous analysis of all cations, anions, and neutrally charged ions.

Multiple CE techniques have been developed by altering the capillary buffer solution. Several of these methods include isoelectric focusing, zone electrophoresis, and electrokinetic chromatography. Let’s first review isoelectric focusing and then move to some of the other techniques.\n\nCapillary isoelectric focusing, also known as CIEF, involves adding ampholytes to the buffer solution in order to create a pH gradient that is used to separate proteins and peptides according to their isoelectric point. The result is a stable gradient that can be maintained.\n\nThe pH gradient is essential in CIEF, and the nature of the pH gradient plays a big role in determining the quality and usefulness of the separation. Two of the most common methods for generating pH gradients involve various types of synthetic buffering molecules, such as carrier ampholytes and synthetic buffer compounds called acrylamide buffers.\n\nCarrier ampholyes are amphoteric electrolytes that carry both current and buffering capacity. They contain both acidic and basic functional groups and form pH gradients under the influence of electric fields. \n\nOn the other hand, acrylamide buffers can be incorporated into polyacrylamide gel matrices that are typically used within the capillary tubing. When used in the correct proportions, these buffers generate immobilized pH gradients under the influence of electric fields.

Also referred to as CZE, capillary zone electrophoresis is the simplest method of CE. A low viscosity buffer fills the capillary, while the analytes in the sample are separated based on their charge-to-mass ratio.

Molecules with a larger ratio, or a smaller size, migrate through the electric field faster and separate first. Very often, capillary electrophoresis refers to the method CZE.

CGE, short for capillary gel electrophoresis, is an adaptation of gel electrophoresis, where slab gels are used to separate molecules based on size and charge, and capillary electrophoresis. The adaptation results in a method that provides scientists with a way to separate molecules entirely on their size.

The general protocol follows four steps:

• The start and end vials, as well as the connecting capillaries, are filled with the gel solution.
• The target sample that will be separated is introduced into the capillary tube.
• An electric field is applied to the sample and its components migrate in the direction opposite of their charge.
• The separated samples are detected using various detection modes, depending on the experiment and the device’s configuration.
• CGE follows the principles of gel electrophoresis, where an electric field is applied through a gel matrix and the molecules, whether they are DNA, RNA, or protein, separate based on their sizes. The larger molecules move slowly through the sieving matrix, while the smaller molecules travel faster. Using capillaries instead of a slab gel provides controlled heat dissipation.

A modified version of CE, this technique involves a surfactant that is added to the buffer solution, in order to form micelles that separate analytes. Micelles are a combination of molecules that form a colloidal particle.

Micellar electrokinetic chromatography (MEKC) uses micelles as a pseudo-stationary partition in order to separate analytes based on their hydrophobic or hydrophilic properties.

This is often used when an analyte contains a neutral molecule that cannot be moved using an electric current. Hydrophobic molecules become trapped within the micelles and travel slower, while hydrophilic molecules travel faster, creating distinct separation.

Capillary electrochromatography, also referred to as CEC, is a hybrid of CZE and HPLC. This technique combines the high-performance of liquid chromatography with the high-efficiency of capillary electrophoresis.

It has unique advantages such as high separation power, selectivity, and sensitivity, while providing short analysis time and low consumption of samples.

This is a fundamental part of capillary electrophoresis. In CE, there are columns within the capillary composed of silica that create a negatively charged inner capillary surface. Cations present in an ionic solution will migrate toward the negatively charged wall, forming an electric double layer. The generation of energy across the column causes cations to migrate towards the cathode and away from the anode.

Electroosmotic flow occurs as the cations gather at the capillary walls, dragging the bulk of the sample towards the cathode. This flow is necessary because it causes movement of nearly all ions present, regardless of charge, in the same direction. This allows for simultaneous analysis of all cations, anions, and neutrally charged ions.