Capillary gel electrophoresis and applications.

Electrophoresis is a separation method in which charged particles migrate under the influence of an electrical field. They migrate under the influence of electric field at various speeds, depending on their charge to mass ratio.

The use of agarose gels in the electrophoresis and use of monolithic column was pioneered by Stellan Hjertén. Tiselius was the first to demonstrate electrophoresis in the free solution to separate proteins without the need for a gel medium. The theoretical principle was laid out by Hjertén. He also demonstrated the free solution zone electrophoresis in small quartz tubes in 1967. Monitors to detect the separation in these small tubes were not available, also the effects of thermal convention are reduced by slowly rotating the tubes around their axis.

Capillary electrophoresis was the main advantage of using only one simple instrument. Capillary electrophoresis has a high voltage power supply, two buffer reservoirs, a capillary and a detector. Basis setup can be modified to enhance the features using autosamplers, multiple injection device, sample/ capillary temperature control, programmable power supply, etc.

Separation in capillary electrophoresis is mainly based on charge to size ratio. By using short separation time high efficiency can be obtained.

Using this standard capillary electrophoresis instrument various different modes of capillary electrophoresis can be carried out. The capillary electrophoresis methods include:

  1. Capillary zone electrophoresis
  2. Capillary gel electrophoresis
  3. Micellar electrokinetic capillary chromatography
  4. Capillary electrochromatograph
  5. Capillary isoelectric focusing
  6. Capillary isotachophoresis

Capillary gel electrophoresis has an excellent detection sensitivity as well as rapid separation time for quantitative and qualitative analysis, thus it is a good alternative to slab gel electrophoresis.

Hjerten and Hjerten and Zhu worked with polyacrylamide filled and agarose filled glass capillaries. They used these capillaries to separate small and large molecules. Another team of Karger and his co-workers worked with gel-filled capillary columns and got extremely high separation efficiency. The capillaries were filled with polyacrylamide gels which also had sodium dodecyl sulfate. This technique is also known as capillary SDS-PAGE electrophoresis. The success in this technique can be partly attributed to procedures developed for crosslinking acrylamide and bisacrylamide monomers inside the fused silica capillaries. Thus, the resulting polyacrylamide was randomly coiled gel structure which is bounded to the capillary walls by addition of a bifunctional reagent. The pore size is adjusted by increasing or decreasing the concentration of gel. As the gel is bound to the capillary the electroosmosis is eliminated.

The main mechanism of separation in capillary gel electrophoresis is based on the difference of the solute size. The solute migrates through the pores of the gel-filled column. Gels are potentially useful mainly because the separation is based on the molecular sieving. Gels also serve as anti-convective media. They also minimize solute diffusion that helps in zone broadening. Gels also prevent solute absorption to the walls of the capillaries and help to eliminate electroosmosis as well. Some of the characteristics that the gels must have is temperature stability and an appropriate pore size.

There are two types of biopolymers used in capillary gel electrophoresis: cross linked and non- crosslinked. In today’s time the matrices are linear non-crosslinked polymers. The most commonly used linear non-crosslinked polymer includes linear polyacrylamide (LPA), polyethylene oxide, poly vinyl alcohol (PVA), polydimethylacrylamide, cellulose derivatives, etc. Precautions should be taken for the elimination of bubble formation during the entire process.

Acrylamide- based sieving polymers in capillary gel electrophoresis

Polyacrylamide based matrix can be used as a linear polyacrylamide or as a crossed linked polyacrylamide gel. The stability of these crosslinked gels can be increased by covalently

attaching these gels to the inner surface of the fused silica capillary. The crosslinked gels only accommodate electrokinetic injections. This leads to a sharp peak due to the inherent sample preconcentration at the interface of the gel and the sample buffer. There is a biased injection of smaller and highly charged molecules into the capillary. When cross linked polymers are used as matrices there can be easy deterioration of the gel structure. In contrast, when we use non-crosslinked polymers they are not affected by high temperature and pH. In addition, the linear non-crosslinked polymers are not attached to the walls of the capillary, so they allow electrokinetic as well as pressure injections. The non-crosslinked polymers pore size an also be varied as the gel is flexible and dynamic.

Agarose as a sieving material in capillary gel electrophoresis

Agarose has a larger pore size as compared to polyacrylamide gels. They are used to separate relatively larger DNA fragments. Agarose doesn’t get cross linked during gelation process. That’s why the inner surface of the capillary must be coated with a suitable noncharged material so that electroosmotic flow can be adequately suppressed. To prepare the agarose gels, required amount of agarose is mixed with the buffer in the reaction mixture. To improve the stability of the gel small amounts of polyalcohol like sorbitol is added. This mixture is heated for 15 minutes degassed and filled in the capillary under pressure.

Polyethylene oxide

It is used in electrophoresis due to its nondenaturing DNA fragment analysis. They also have self-coating properties.

Polyvinylpyrrolidone sieving

It useful for the rapid molecular diagnostic capable of detecting common mutations using primer extension techniques combined with capillary electrophoresis.

Polysaccharides as sieving material

Polyethylene glycol and dextran are used due to their low UV absorption.

Principle capillary gel electrophoresis

  • Electrophoretic mobility

The electrical force (Fe) is the product of the electric filed (E) and the net charge of the analyte molecule (Q).

Fe = QE.

The polymer matrix in the capillary tubing also produces a frictional force in the opposite direction. When the electric field is applied, the charged analyte starts moving according to the Newton’s second law of motion. The analyte molecule migrates with a stead state velocity. Different electrophoretic mobilities are due to the different shapes, sizes or net charge of the analyte, which influences the electromigration. In capillary gel electrophoresis, molecular sieving is explained by using the Ogston theory. When the hydrodynamic radius of the analyte molecule is in the same range as that of the pore size of the sieving matrix. The retardation coefficient depends on the molecular weight of the molecule at constant polymer concentration. Large biomolecules containing flexible chains can migrate through the pores of these polymers networks which have a smaller size that the analyte. This is done by using the ‘snake like’ motion when migrating through the small pores. Column efficiency and resolution

The resolution of the column is measured as the difference between the electrophoretic mobilities of the analyte of interest. Temperature should the maintained to achieve adequate migration. As the limiting factor of the high resolution is the so-called Joule heat which is produced by the applied power.

Instrumentation

A typical instrument used for capillary gel electrophoresis consist of a separation capillary, high voltage power supply and a detection assembly. The length of the capillary varies from 10-100 cm. Either bare fused silica or coated capillaries can be used in a single or multicapillary system. The inner surface of the capillary should be coated to suppress the electroosmotic flow. The coating is done by using a bifunctional reagent gama- methacryloxypropyl-trimethoxysilane and subsequent cross-linking the surface bound methhylacryl groups with linear polyacrylamide. The coating also prevents

adsorption of the analyte molecule. Electrokinetic injections are mostly used to introduce the sample as the high viscosity of the media prevents pressure injections. Electrokinetic injections use’s electric field to force it’s charged analyte molecules across the gel. During this injection process, the electrodes and both the ends of the capillaries are immersed into the samples respectively and outlet buffer vials. The applied voltage or pressure drives the analyte molecules into the capillary tube. As the separation process starts the components migrate through the gel -buffer system filled capillary tube. The detection can be carried out using various detectors.

Detectors

Various detectors can be used to detect the end result of the capillary gel electrophoresis.

  1. UV- Visible detector

The light sources in these detectors can be either single line or continuous source. Atomic lamps eg. Hg lamp, Cd lamp, Zn lamp and As lamp can be used to generate strong emission lines and well-defined wavelengths. For continuous sources deuterium and tungsten-halogen lamps can be used.

  • Diode Array detectors

Diode array detectors are a Multi-wavelenght detector, which utilizes a “reverse optics” design. A diode array detector provides spectral analysis of each sample component along with qualitative information.

  • Laser induced fluorescence detector.

It is the most sensitive online detection scheme which is even capable of single molecule detection. It requires chemical derivatization. Various dyes and reagents are used for detection carbohydrates, proteins, nucleic acids, etc

Light- Emitting Diode -Induced Fluorescence detection.

When we use fluorescence detection, the detection should have high sensitivity and a strong light source. Now a days LEDIF detection has been used. In this new generation LEDs offer an alternative light source to laser for fluorescent detection.

Advantages:

  1. Capillary gel electrophoresis has the potential advantage of using nanogram sample capabilities.
  2. It can be automated easily.
  3. Accurate quantitation.
  4. It is highly sensitive.
  5. It has higher separation efficiency.

Limitations:

  1. They lack preparative capabilities and a vulnerable to gel-filled column damage.
  2. Neutral molecules would do migrate through the gel as the electroosmotic flow in this operation is suppressed.

Application:

  • Cross linked polyacrylamide gels can be used to separate shorter single stranded DNA and small proteins as well using SDS- based CGE.
  • The linear polyacrylamide coated capillaries can be used to separate double stranded DNA, SDS- protein complexes as well as complex carbohydrates.
  • Agarose gel capillaries can be used to analyze relatively larger DNA fragments.
  • In the biotechnology industry, capillary SDS gel electrophoresis is used for the analysis of therapeutic recombinant monoclonal antibody (rmAb).
  • Capillary SDS gel electrophoresis is also used for the characterization of human colon cancer adenocarcinoma cells.
  • It is also used for manufacturing of protein pharmaceuticals in bulk.
  • The SDS gel electrophoresis has been used for protein characterization of bacterial lysate and even in profiling of human serum protein.
  • Capillary gel electrophoresis can be used for rapid and efficient analysis of biopolymers.
  • Capillary gel electrophoresis can be used to characterize Clostridium difficile. It divides 24 isolates belonging to PCR ribotyping type 014 into seven groups.
  • Gene amplified on polydimethylsiloxane- glass hybrid microchip can be analyzed by using capillary gel electrophoresis.
  • Capillary gel electrophoresis can be used for the diagnosis of the aldehyde dehydrogenase 2 genotype. Capillary gel electrophoresis provides a high speed, high resolution analytical tool for determining genetic type.
  • Capillary gel electrophoresis was used in the Human genome project. They used the linear noncross- linked polymer matrices and even used the high pressure injection method. This help in an automated largescale DNA sequencing.
  • The determination and quantification of endogenous beta-galactosidase in the crude E. coli lysate can be done by immumoprecipitation combined with microchip capillary gel electrophoresis with laser- induced fluorescence.
  • Capillary gel electrophoresis is used to group L. delbrueckii ssp bulgaris in a cluster. L. delbrueckii ssp. lactis and L. delbrueckii spp. delbrueckii into another. Separation is done using fused silica capillary column. By using capillary gel electrophoresis, the whole cell protein can be distinguished into the taxonomic status.
  • Capillary gel electrophoresis can also be used in monitoring cloning procedures. As it highly sensitive and resolution. It is also useful to detect manipulation with only small difference in fragment length. The size, concentration and purity of the DNA simultaneously.