Size exclusion chromatography

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Size-exclusion chromatography (SEC) is a chromatographic method in which molecules in solution are separated by their size, not by molecular weight. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel-filtration chromatography, versus the name Gel permeation chromatography, which is used when an organic solvent is used as a mobile phase. SEC is a widely used polymer characterization method because of its ability to provide good Mw results for polymers.

Contents

Applications

The main application of gel-filtration chromatography is the fractionation of proteins and other water-soluble polymers, while gel permeation chromatography is used to analyze the molecular weight distribution of organic-soluble polymers. Either technique should not be confused with gel electrophoresis, where an electric field is used to "pull" or "push" molecules through the gel depending on their electrical charges.

SEC is a widely used technique for the purification and analysis of synthetic and biological polymers, such as proteins, polysaccharides and nucleic acids. Biologists and biochemists typically use a gel medium — usually polyacrylamide, dextran or agarose — and filter under low pressure. Polymer chemists typically use either a silica or crosslinked polystyrene medium under a higher pressure. These media are known as the stationary phase.

Advantages

The advantages of this method include good separation of large molecules from the small molecules with a minimal volume of eluate,[1] and that various solutions can be applied without interfering with the filtration process, all while preserving the biological activity of the particles to be separated. The technique is generally combined with others that further separate molecules by other characteristics, such as acidity, basicity, charge, and affinity for certain compounds. With size exclusion chromatography, there are short and well-defined separation times and narrow bands, which lead to good sensitivity. There is also no sample loss because solutes do not interact with the stationary phase. Disadvantages are, for example, that only a limited number of bands can be accommodated because the time scale of the chromatogram is short, and, in general, there has to be a 10% difference in molecular mass to have a good resolution[2]

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