Name: size-exclusion chromatography in protein analysis
Uploaded: 2024-12-05T18:32:00.000Z
Duration: 1 min 8 s
Channel: JoVE

Size-exclusion chromatography, or SEC, involves separating molecules based on size.
Here, the stationary phase can be silica particles or cross-linked polymer resin beads that contain a network of uniform pores.
Large molecules are excluded from entering the pores and eluted first, whereas very small molecules are retained the longest and eluted last.
Intermediate-sized molecules are separated in the retention window and undergo fractionation.
The retention time of the molecules is directly influenced by their size.
The experimental calibration curve obtained using standard substances is then used to estimate the unknown molecular mass.
Size exclusion chromatography using hydrophilic packing is known as gel filtration and is applied to water-soluble samples. Hydrophobic packing materials are used in gel permeation chromatography for substances soluble in less-polar organic solvents.
Both these methods are useful for the separation of polymers or macromolecules.

size-exclusion chromatography

Size-Exclusion Chromatography

In size-exclusion chromatography (SEC), also known as molecular-exclusion or gel-permeation chromatography, molecules are separated based on their sizes. This technique is important for separating large molecules such as polymers and biomolecules. The two classes of micron-sized stationary phases encountered in SEC are silica particles and cross-linked polymer resin beads. Both materials are porous, but their pore sizes vary significantly.
Silica particles offer advantages such as rigidity, stability, and compatibility with various solvents. In contrast, polymer beads come in different pore sizes and can be hydrophilic (water-loving) or hydrophobic (water-repelling). Hydrophilic gels like agar and polyacrylamide are used for aqueous separations, while hydrophobic gels such as polystyrene-divinylbenzene are preferred for non-polar solvents. Silica particles used for size exclusion are deactivated to prevent unwanted interactions, while polymer resins are synthesized without exchange sites.
Smaller solutes spend more time within the pores of the stationary phase, resulting in a longer elution time from the column. The retention volume for a solute depends on its distribution ratio, which ranges from 0 at the exclusion limit to 1 at the inclusion limit. This reflects how well the solute penetrates the pores.
SEC is a notable application in the analysis of protein mixtures and the determination of formula weights. Calibration curves can be prepared between the exclusion and inclusion limits, but accurate formula weight determinations require carefully chosen standards to minimize the effect of shape.
This technique can be carried out using conventional HPLC instrumentation by replacing the HPLC column with an appropriate size-exclusion column. A UV/Vis detector is typically used to obtain chromatograms.

In size-exclusion chromatography (SEC), also known as molecular-exclusion or gel-permeation chromatography, molecules are separated based on their sizes. ...

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JoVE Core

Analytical Chemistry

Unit 1

Principles of Chromatography

KEY TERMS AND CONCEPTS

size-exclusion chromatography in protein analysis

Size-exclusion chromatography separates molecules by size, and it is often used for large molecules such as polymers and biomolecules. It is also called molecular-exclusion chromatography or gel-permeation chromatography. In this method, the stationary phase is made of micron-sized particles or resin beads that contain pores.

The two main stationary phases are silica particles and cross-linked polymer resin beads. Both are porous, but their pore sizes can differ a lot. Silica particles are rigid, stable, and compatible with many solvents. They are also deactivated so they do not interact in unwanted ways with the sample.

Polymer beads are made with different pore sizes and can be hydrophilic or hydrophobic. Hydrophilic gels such as agar and polyacrylamide are used for aqueous separations. Hydrophobic gels such as polystyrene-divinylbenzene are preferred for non-polar solvents. Polymer resins are synthesized without exchange sites.

Smaller solutes spend more time inside the pores of the stationary phase. As a result, they leave the column later. The retention volume depends on the distribution ratio, which ranges from 0 at the exclusion limit to 1 at the inclusion limit. This shows how well a solute can enter the pores.

SEC is useful for analyzing protein mixtures and for determining formula weights. Calibration curves can be made between the exclusion and inclusion limits. For accurate formula weight results, the standards must be chosen carefully so shape has less effect. The technique can be run on standard HPLC equipment by swapping in a size-exclusion column, and a UV/Vis detector is usually used to record the chromatogram.

Size-Exclusion Chromatography in Protein Analysis

size-exclusion-chromatography-protein-analysis

chromatographic methods: terminology

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Size-exclusion chromatography separates molecules by size, and it is often used for large molecules such as polymers and biomolecules. It is also called molecular-exclusion chromatography or gel-permeation chromatography. In this method, the stationary phase is made of micron-sized particles or resin beads that contain pores. The two main stationary phases are silica particles and cross-linked polymer resin beads. Both are porous, but their pore sizes can differ a lot. Silica particles are...

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capillary electrophoresis: instrumentation

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key terms and concepts

Learn how size-exclusion chromatography separates molecules by size, using porous silica or polymer beads to analyze proteins and estimate formula weights.

Size-Exclusion Chromatography in Protein Analysis | JoVE K12

analytical chemistry

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Size-Exclusion Chromatography in Protein Analysis

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