Capillary electrophoresis how does it work

From research to single-gene to small gene panels, CE can help achieve research goals across a diverse set of applications. Don't have an account? Create Account. Sign in Quick Order. Search Thermo Fisher Scientific. Search All. What are the applications of capillary electrophoresis?

See Navigation. Some of the advantages of CE compared to conventional polyacrylamide gel electrophoresis include: Ease of use—no gels to pour Reusability—the polymer matrix can be reused product-dependent Fast separation times Better resolution single base pair Automated reads—optical sequence reading Higher throughput—multiple capillaries can be used simultaneously Complete automation—a complete workflow including CE is available on automatic genetic analysis systems CE enables a wide variety of applications for a broad range of research areas Figure 1.

Click image to enlarge Figure 1. Sanger sequencing by CE is best suited for single genes, single-sample variant detection, or next-generation sequencing NGS verification. Figure 2. Sanger sequencing workflow. The Sanger sequencing workflow enables chain-terminated, end-labeled fragments to be generated and subsequently read as a sequence.

Figure 3. Fragment analysis by CE workflow. Capillaries were used after the s. 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. Generally, the charged species begin to move in electric fields. The rate of the electroosmotic flow is governed by the following equation:.

Because the electrophoretic mobility is greater than the electroosmotic flow, negatively charged particles, which are naturally attracted to the positively charged anode, will separate out as well. The EOF works best with a large zeta potential between the cation layers, a large diffuse layer of cations to drag more molecules towards the cathode, low resistance from the surrounding solution, and buffer with pH of 9 so that all the SiOH groups are ionized.

They can be classified into continuous and discontinuous systems as shown in Figure 3. A continuous system has a background electrolyte acting throughout the capillary as a buffer. This can be broken down into kinetic constant electrolyte composition and steady-state varying electrolyte composition processes.

A discontinuous system keeps the sample in distinct zones separated by two different electrolytes. Capillary Zone Electrophoresis CZE , also known as free solution capillary electrophoresis, it is the most commonly used technique of the six methods. A mixture in a solution can be separated into its individual components quickly and easily. The separation is based on the differences in electrophoretic mobility, which is directed proportional to the charge on the molecule, and inversely proportional to the viscosity of the solvent and radius of the atom.

The velocity at which the ion moves is directly proportional to the electrophoretic mobility and the magnitude of the electric field. T he fused silica capillaries have silanol groups that become ionized in the buffer. The negatively charged SiO - ions attract positively charged cations, which form two layers—a stationary and diffuse cation layer. Anions in solution are attracted to the positively charged anode, but get swept to the cathode as well. Cations with the largest charge-to-mass ratios separate out first, followed by cations with reduced ratios, neutral species, anions with smaller charge-to-mass ratios, and finally anions with greater ratios.

The electroosmotic velocity can be adjusted by altering pH, the viscosity of the solvent, ionic strength, voltage, and the dielectric constant of the buffer. CGE uses separation based on the difference in solute size as the particles migrate through the gel.

Gels are useful because they minimize solute diffusion that causes zone broadening, prevent the capillary walls from absorbing the solute, and limit the heat transfer by slowing down the molecules. It is a highly sensitive system and only requires a small amount of sample. MEKC is a separation technique that is based on solutes partitioning between micelles and the solvent.

Micelles are aggregates of surfactant molecules that form when a surfactant is added to a solution above the critical micelle concentration. The aggregates have polar negatively charged surfaces and are naturally attracted to the positively charged anode. Because of the electroosmotic flow toward the cathode, the micelles are pulled to the cathode as well, but at a slower rate. Hydrophobic molecules will spend the majority of their time in the micelle, while hydrophilic molecules will migrate quicker through the solvent.

DNA fragments were separated based on size, with a resolution down to 1 base pair. This makes sequencing fragments of DNA possible, along with determining other parameters like copy number variants, which is used to diagnose potential genetic diseases. A protein can be modified by various functional groups that are chemically attached to different locations.

Different copies of the same protein can vary with different modifications, which will change the charge and size of each protein. Running the purified proteins through a CE that is attached to a mass spectrometer can separate proteins based on which modifications are present, and also identify the type and location of the modification. You've just watched JoVE's introduction to capillary electrophoresis.

You should now understand how CE separates molecules based on charge and mass, and how to run a sample on the CE in the lab. Electropherograms collected for diet Pepsi and Pepsi samples are shown in Figures 1 and 2 , respectively. The three peaks for caffeine, aspartame, and benzoic acid are observed in diet Pepsi and have similar migration times as the standards. For regular Pepsi, the caffeine peak is present but not the aspartame and benzoic acid peaks.

The CE analysis is fast as the migration times are only 3—4 min. The calibration curve for caffeine is shown in Figure 3. This curve can be used to calculate the concentration of caffeine in each sample. Figure 1. CE analysis of Diet Pepsi. The red are standards of caffeine, aspartame, and benzoic acid. The black is a diet Pepsi sample. Please click here to view a larger version of this figure.

Figure 2. CE analysis of Pepsi. The black is a Pepsi sample while the red is a sample of standards of caffeine, aspartame, and benzoic acid. There is no aspartame or benzoic acid, indicating the soda is not diet. Figure 3. Caffeine calibration plot with CE. A plot of the peak area vs concentration for caffeine standards measured with CE.

Capillary electrophoresis is used for many specialty separations. For example, it is used in the pharmaceutical industry for quality testing, to make sure there are no side products or interferents. CE is particularly useful for separating drugs with a basic amino group, as the walls of the capillary can be made neutral with an acidic pH and thus the drug will not stick to the capillary.

This mode of CE is capillary gel electrophoresis and for these separations, a polymer is injected into the CE capillary. The polymer gives an additional mode of separation based on size, as the smaller fragments can travel faster through the gel. This is called sieving, and along with the electrophoretic separation, it has 1 base pair resolution for DNA analysis. Analytical Chemistry. Capillary Electrophoresis CE. To learn more about our GDPR policies click here. If you want more info regarding data storage, please contact gdpr jove.

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Sign in or start your free trial. Previous Video Next Video. Overview Source: Laboratory of Dr. Jill Venton - University of Virginia Capillary electrophoresis CE is a separation technique that separates molecules in an electric field according to size and charge.

Log in or Start trial to access full content. Using the computer software, turn on the light source for UV analysis to allow it to warm up.

Some software has an indicator when the lamp is ready for use lamp icon turns color. Make a methods file. Set the important parameters for running the CE.