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How does size-exclusion chromatography separate molecules by size?
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Biochemistry
In biochemistry, researchers study the chemical substances and processes that occur in living organisms and cells. In particular, various processes by which cells gain energy, such as epinephrine-stimulated cAMP synthesis, have been deduced through the study of biochemistry.
Size-exclusion chromatography (SEC) separates molecules based on their physical size by utilizing a stationary phase composed of a porous matrix.12
The separation process functions as follows:
- Pore Penetration: As a mixture moves through the chromatographic column, solute molecules diffuse into and out of the pores within the stationary phase. The depth of this penetration is determined by the size of the molecule.2
- Differential Migration: Smaller molecules can enter more of the available pores, which increases the path they must travel and slows their progress through the column. Conversely, larger molecules are unable to enter many of the pores and are effectively excluded, allowing them to flow through the system more rapidly.132
- Elution Order: Because of these differences in migration, the largest molecules emerge from the column first, while smaller molecules appear later.12
This technique is widely used in biochemistry for the analysis and purification of proteins, polymers, and other large biological molecules.13