User:Chem4066sp13/sandbox cf

Capillary Gel Electrophoresis (CGE) is an adaptation of traditional gel electrophoresis which uses polymers to create a molecular sieve, and separates species based on their charge to mass ratio in a capillary tube. Capillary gel electrophoresis separates species by differences in solute size by implementing a polymeric material inside the capillary. Using gels is particularly advantageous because they can minimize solute diffusion causing zone broadening, prevent capillary walls from absorbing the solute, and limit heat transfer. Capillary gel electrophoresis was introduced in the 1980's as a more efficient way to separate proteins. The most widely used CGE technique is sodium dodecyl sulfate polyacrylamide gel electrophoresis.

=Methodology=

Instrumentation
The apparatus for capillary gel electrophoresis is identical to capillary electrophoresis which consists of a capillary column, on-column detector, and a high voltage power supply as well as a data output and handling device. The only difference is the separation media, where capillary gel electrophoresis uses a sieving matrix and capillary electrophoresis uses a background electrolyte solution.



Separation Theory
Addition of a sieving matrix to the capillary aids in the separation of the analytes by separating them by size. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. The addition of a sieving matrix suppresses the electroosmotic flow (EOF). There are two theories of separation: ogsten sieving and reptation. Ogsten sieving occurs when the molecules retain there standard shape but does not account for molecules stretching or deforming to squeeze through the pores of the sieving matrix. Reptation is when the molecule moves through the separation media as a snake. Reptation typically occurs at higher field strength and mobility becomes independent of molecular weight.



Sieving Matrices
There are many different sieving matrices that are implemented in CGE and each is specific to a type of separation. Polyacrylamide gel is widely used to separate proteins. In addition, various polysaccharides can also be used to separate proteins. Methyl cellulose, a polymer consisting of numerous linked glucose molecules, is often used to separate DNA fragments. The concentration of the sieving matrix can be adjusted to yield better resolution between analytes, but increased gel concentration decresases the molecular weight range accessible within the run.

Detection
The typical detection mode in capillary gel electrophoresis is UV absorption because most analytes absorb light in the UV range. Sometimes LIF (laser induced fluorescence) detectors are implemented to improve sensitivity, but this requires the analytes to sometimes be fluorescently labeled. Mass spectrometry is rarely linked to CGE because the sieving matrices interfere.

Efficiency and Resolution
Capillary gel electrophoresis was implemented to replace gel electrophoresis as a faster analytical tool. CGE can be up to twenty-five times faster than gel electrophoresis, it has higher resolution and it requires smaller sample loads. Additionally, CGE instrumentation allows for automation and highly multiplexed operation.

=History=

Capillary gel electrophoresis was first published in the 1980's and used agarose and cross-linked polyacrylamide (CPA) as sieving matrices which were prepared directly in the column. In the early 1990's, linear polyacrylamide (LPA) was introduced to replace CPA. However,the lifetimes of these columns was poor and was usually limited to less than 10 runs. Currently, replaceable and water-soluble linear or slightly branched polymers are used as sieving matrices for capillary gel electrophoresis.

=Applications=


 * Detecting Proteins in Biological Fluids- Capillary gel electrophoresis is a powerful tool in analyzing proteins in biological fluids. These fluids are normally very difficult to analyze due to the complexity of sample media.


 * Detecting Proteins in Food Products- Monitoring food safety regulations is becoming increasingly important CGE is a primary method for analyzing a food sample. With CGE it is possible to separate and identify different proteins contained in a meat sample. This is useful because meat quality can be indicated by the profile and quantity of water-soluble and salt-soluble proteins. A similar analysis can be done in cow's milk by analyzing cider proteins and determining their relative molecular masses.


 * Agricultural Product Analysis- The soluble-protein RuBisCo accounts for more than 50% of the protein content in Chloroplasts, and is a key enzyme in the photosynthetic fixation of Carbon Dioxide. By measuring the content RuBisCo it is possible to get an accurate indication of a plants physiological status.


 * Human Genome Project- Capillary gel electrophoresis has been recently been implemented to help aid in the sequencing of the human genome. Capillary gel electrophoresis is a very fast technique and through multiplexing of the instrumentation, the human genome sequencing rate has increased exponentially.


 * Biopharmaceutical industry- Capillary gel electrophoresis has become an important tool to support analytical characterization, process development, and quality control of therapeutic recombinant monoclonal antibodies (rMAbs).

=See also=


 * Capillary electrophoresis
 * Gel electrophoresis
 * Micellar electrokinetic chromatography

=References=