Size exclusion chromatography (SEC) separates molecules in a mixture based purely on their size in solution. This method, sometimes called gel filtration chromatography when used with aqueous solvents for biological samples, fractionates components according to their hydrodynamic radius, which is closely related to molecular weight. SEC is widely employed across various scientific disciplines, including biochemistry for handling proteins and nucleic acids, and polymer science for characterizing synthetic materials. The technique is gentle because it avoids strong chemical interactions that could alter the integrity of sensitive molecules.
The Principle of Size-Based Separation
The fundamental operation of size exclusion chromatography relies on a stationary phase packed inside a column that acts as a molecular sieve. This stationary phase consists of spherical, porous beads, typically made from materials like cross-linked agarose or dextran, which contain a vast network of uniform pores. A mobile phase, usually a buffer or solvent, continuously flows through the column, carrying the sample mixture.
When the sample is introduced, the path of the molecules is determined by their physical size relative to the pores in the beads. Molecules significantly larger than the maximum pore size are completely excluded from entering the porous network. These large molecules are forced to flow around the beads in the interstitial volume. Because they traverse only the space between the beads, they move quickly and are the first components to exit the column.
Conversely, molecules that are very small can freely penetrate almost all the pores within the stationary phase beads. These smaller components encounter a much longer, more tortuous pathway as they diffuse in and out of the pores, effectively increasing the distance they must travel before exiting the column. Consequently, small molecules are retained for the longest time and elute last from the column. Molecules of intermediate size penetrate the pores to varying degrees, with their retention time falling between the largest and smallest molecules.
Molecules elute in order of decreasing size. The largest molecular size that is completely excluded from the pores defines the exclusion limit of the column. Any molecule larger than this limit will elute at the same time, unable to be separated from one another. The stationary phase material is selected with a specific pore size distribution to ensure optimal separation across the molecular weight range of interest.
Essential Components of the SEC System
A functional size exclusion chromatography system requires several specialized hardware components working together to achieve the separation and analysis. At the core of the system is the chromatography column, a cylindrical tube tightly packed with the porous resin beads that serve as the stationary phase. The specific dimensions of the column, such as its length and diameter, are selected based on whether the goal is high-resolution analytical separation or larger-scale preparative purification.
A high-precision pump drives the mobile phase, a solvent or buffer, through the column. The mobile phase must be chemically inert and dissolve the sample without causing interaction with the stationary phase. The sample mixture is introduced into the flowing mobile phase via an injection system. This system ensures the sample enters the column as a narrow, compact band to maximize separation efficiency.
As the separated components exit the column, they flow directly into a detector. The detector continuously monitors the eluent, typically by measuring a physical property of the molecules, such as ultraviolet light absorbance. This measurement generates a chromatogram, which plots the detector signal against the elution volume. Each peak on the chromatogram corresponds to a distinct group of molecules separated by size, with its position indicating the elution time and size.
Primary Applications of Size Exclusion Chromatography
One primary application of SEC is determining the molecular weight distribution (MWD) of synthetic polymers. By separating a polymer sample into its constituent chain lengths, researchers gain a detailed understanding of the polymer’s quality and physical properties. These properties are heavily dependent on the mix of different molecular weights present.
In biochemistry, SEC is widely used for the purification of large biomolecules, such as proteins and antibodies. It is frequently the final step in a multi-stage purification scheme, providing a high-resolution separation to isolate the target molecule from any remaining contaminants or unwanted aggregates. The technique is particularly effective at separating the desired monomeric protein from larger species like dimers or oligomers, which are often inactive or undesirable for therapeutic use.
A common application of SEC is desalting or buffer exchange, which quickly removes small molecules like salts, detergents, or free labels from a solution containing much larger biomolecules. In this mode, the large molecules are completely excluded from the pores and elute rapidly, while the small salt ions are retained. This process is often performed before downstream analytical techniques that are sensitive to high salt concentrations. Furthermore, SEC plays a role in quality control, where it is used to assess the stability of biopharmaceutical products by monitoring for the formation of size-related degradation products or aggregates over time.