Size exclusion chromatography

Size Exclusion Chromatography (SEC), also known as Gel Filtration Chromatography, is a chromatographic method that separates molecules based on their size. It is widely used in the biochemistry and biotechnology fields for the analysis and purification of proteins, polymers, and other macromolecules.

Principle

Size exclusion chromatography operates on the principle of molecular sieving. The stationary phase consists of porous beads, typically made of agarose or polyacrylamide, which have a defined pore size. As a sample is passed through the column, smaller molecules enter the pores and are delayed, while larger molecules are excluded from the pores and elute earlier.

Components

Stationary Phase

The stationary phase in SEC is composed of porous beads. These beads are made from materials such as dextran, agarose, or polyacrylamide. The choice of material and pore size depends on the size range of the molecules to be separated.

Mobile Phase

The mobile phase in SEC is typically an aqueous buffer. The choice of buffer depends on the stability and solubility of the sample molecules. Common buffers include phosphate-buffered saline and Tris buffer.

Column

SEC columns are typically long and narrow to provide sufficient resolution. The column is packed with the stationary phase beads and equilibrated with the mobile phase before sample introduction.

Applications

Protein Purification

SEC is extensively used for the purification of proteins. It allows for the separation of proteins based on their size, which is useful for removing aggregates or separating monomers from dimers and higher-order oligomers.

Polymer Analysis

In polymer chemistry, SEC is used to determine the molecular weight distribution of polymers. This information is crucial for understanding the properties and behavior of polymer materials.

Biopharmaceuticals

SEC is employed in the development and quality control of biopharmaceuticals, such as monoclonal antibodies, to ensure product consistency and purity.

Advantages

- Non-destructive: SEC does not alter the sample, making it ideal for sensitive molecules. - Simple preparation: Minimal sample preparation is required. - Versatile: Can be used for a wide range of molecules, from small peptides to large protein complexes.

Limitations

- Resolution: Limited resolution compared to other chromatographic techniques like high-performance liquid chromatography (HPLC). - Sample Dilution: Samples can become diluted during the separation process. - Limited Range: Effective only for molecules within a certain size range.

See Also

• Chromatography
• Gel Electrophoresis
• Affinity Chromatography
• Ion Exchange Chromatography

External Links

• [Link to educational resources on chromatography]
• [Link to chromatography equipment manufacturers]