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Capillary Electrophoresis Equipment

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

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Capillary electrophoresis equipment

Capillary electrophoresis (CE) is an electrophoretic technique for separating and identifying charged molecules, such as amino acids, peptides, nucleic acids, and proteins.

Biotech diagram

It can also be used to separate various drugs based on their rate of migration through a given medium.

CE is part of a family of related techniques that use very narrow capillaries and buffers to perform high-efficiency electrophoretic separation of both small and large molecules. The separations occur through the use of high voltages, which creates electroosmotic flow (EOF) within the buffer solutions and ions inside the capillary tubing.

EOF is considered the driving force behind capillary electrophoresis, and results in the simultaneous analysis of anions, cations, and neutral charged molecules in a single analysis.

CE shares characteristics with traditional polyacrylamide gel electrophoresis (PAGE) and high-performance liquid chromatography (HPLC). In fact, CE and gel electrophoresis (GE) are considered to be in the same family. But, CE is generally seen as the faster method, providing higher throughput and resolution because the thin tubing used offer a higher surface-to-volume ratio than gels.

In contrast to the slab gels used in GE, capillary electrophoresis systems and instruments utilize these thin capillary tubes, or silica capillaries, to speed up the electrophoretic process. The tube can dissipate heat more quickly as well, allowing for higher voltages to run without any risk of overheating.

The high-voltage environment increases the velocity at which a molecule passes through the capillary, shortening the separation process and making CE highly efficient.

As well, capillary electrophoresis uses modern data acquisition devices to observe the ions as they pass through the capillary past the detector. The signal is recorded and sent to a computer with software for digital collection and analysis. The device requires very little amounts of a sample to work, so you can potentially save more money when using high-cost materials.

Furthermore, many capillary electrophoresis instruments are automated, consume limited amounts of reagents, offer precise quantitative analysis, and process a larger variety of analytes in comparison to other separation techniques. The automation is seen as advantageous to any laboratory that works with high-cost materials and is concerned with efficiency and high-resolution.

Below are the basic CE instrument components, as well as the common applications of capillary electrophoresis.

Components, Flow methods, & Electrophoresis Techniques

An open hand with a DNA strand coming out of the palm

CE systems and instruments are used in a variety of fields and industries, including biomedicine, forensic sciences, chemistry, and pharmaceuticals. In biomedicine, CE has been extremely useful tool for separating and characterizing synthetic polymers, specifically biopolymers over the past two decades.

However, CE has perhaps had the greatest impact in clinical laboratories, helping with applications including:

  • Protein analysis of proteins in serums, urine, and body fluids
  • Hemoglobin variant analysis
  • Molecular diagnostics
  • Lipoprotein analysis
  • Forensic and therapeutic drug screening

CE Instrument Components

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:

Electroosmotic Flow (EOF)

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.

Isoelectric Focusing

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.

Zone Electrophoresis

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.

Capillary Gel Electrophoresis

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.

Micellar Electrokinetic Chromatography

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.

Electrochromatography

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.

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