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An Introduction to Size Exclusion Chromatography / Gel Permeation Chromatography in 30 Minutes

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By submitting a product review you are not only sharing your experiences with others, but also gaining helpful insight from others in the research community. In this way, molecules in a mixed sample, such as a polydisperse polymer or a mixture of protein molecules, are organized according to their size.

More Detailed Look at GPC

After the separation, one or more detectors is used to characterize the sample. This always involves a concentration detector and may or may not include advanced detectors, such as light scattering and intrinsic viscosity. The primary goal of this characterization is to measure the molecular weight and molecular weight distribution but may also include the measurement of other properties such as size and structure.

This Paper Offers a Comprehensive Educational Insight into the GPC/SEC Technology and Applications

The bulk physical properties, such as strength and toughness, of synthetic and natural polymers, or the activity and efficacy of proteins and biopharmaceuticals are strongly dependent on their molecular properties. In a large number of industries, there is a clear and strong desire to control molecular weight and structure in order to better manipulate and control the bulk properties of the material. For example, in polymer chemistry, different molecular parameters can have effects on different bulk properties. For proteins, molecular weight, and therefore, oligomeric state directly relates to their activity either as a biopharmaceutical, or in an assay.

The amount and size of any aggregates present in a sample will result in a loss of sample activity and in the case of biopharmaceuticals, can also stimulate an immune response affecting the efficacy and safety of such drugs. A separation technique in which separation, mainly according to the hydrodynamic volume of the molecules or particles, takes place in a porous non-adsorbing material with pores of approximately the same size as the effective dimensions in solution of the molecules to be separated.

As SEC covers such a broad application range, the main focus areas are slightly different between applications. For synthetic and natural polymers the main purpose of the technique is typically for the determination of molecular mass averages and molecular mass distribution of the sample.

Gel permeation chromatography/ Size exclusion chromatography

For proteins, the main focus is typically the determination of monomeric and oligomeric states and their quantification. Further information, such as size, structural and compositional information, can be derived from multi-detector SEC-systems.

Topic 4.9 - Size exclusion chromatography

In addition to use at analytical level, SEC can also be applied at a preparative scale for large-scale separations or purification purposes whereby eluting molecular weight fractions can be collected in a suitable container and isolated. SEC can also be applied to solid matter nanoparticles dispersed in a liquid.

Despite the increasing interest in nanoparticle characterization, SEC studies on nanoparticles still form only a minor area of interest and the main focus of SEC remains on characterization on a molecular level. SEC is a liquid chromatography LC method, a subset of HPLC, and requires the sample material to be completely dissolved with the individual molecules dispersed and not interacting. In certain cases, where the aim is to study assemblies of molecules, e.

Although dissolution is straightforward for a lot of samples, care has to be taken to make sure samples do go into solution completely and do not degrade or get modified by the dissolution process. The columns used for SEC are filled with a gel matrix containing highly porous spherical particles.

Size Exclusion Chromatography / Gel Permeation Chromatography: An Introduction in 30 Minutes

The most common materials used in these gels include cross-linked polymers like polystyrene, acrylates, dextran or silica. A constant eluent flow is forced through the column by an isocratic pump. The sample solution is introduced into the flow by means of an injection valve and loop. Under ideal conditions, there is no interaction between the sample and the gel, meaning that the separation process should be based purely on diffusion of the analyte while the solution travels through the stationary phase.

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