Gelfiltration

Gel filtration, also known as size-exclusion chromatography (SEC), is a chromatographic method that separates solutes based on their size as they pass through a porous gel matrix in a column. The gel consists of cross-linked polymer beads with a distribution of pore sizes, creating a network that differentiates molecules by how readily they can enter the pores.

During elution, large molecules are excluded from many pores and travel through the column in or near the void volume, eluting first. Smaller molecules penetrate more pores and take longer, eluting later. The elution volume correlates with size or hydrodynamic radius, and measurements are typically interpreted using a calibration curve generated with standards of known molecular weight or radius.

Common applications include desalting or buffer exchange, removal of small molecules from samples, purification of biomolecules that do not interact strongly with the matrix, and estimation of molecular weight. Gel filtration is frequently used to separate proteins, nucleic acids, polysaccharides, and protein complexes, as well as to resolve aggregates from monomeric species.

Materials and practical considerations: Gel matrices include dextran-based gels (such as Sephadex), agarose-based gels (such as Sepharose), and polyacrylamide-based gels (such as Bio-Gel). Columns are operated in aqueous buffers under moderate conditions, with selectivity and performance dependent on minimizing non-size interactions between analytes and the matrix. Choosing a column with an appropriate fractionation range is crucial for efficient separation, and gel filtration is generally gentle and non-denaturing but can cause sample dilution.

Limitations include limited resolution for molecules of similar size, potential distortions if molecules interact with the matrix, and dilution of the sample; it is typically used in conjunction with other purification methods.