Gel filtration chromatography (GFC) is a widely used laboratory technique that separates molecules primarily based on their size. It is particularly useful for large biological molecules, such as proteins and nucleic acids, as it separates them without altering their natural structure or activity. The technique is also known as size exclusion chromatography (SEC) or molecular sieve chromatography.
How Molecules Separate by Size
GFC relies on the differential movement of molecules through a porous stationary phase. This stationary phase consists of microscopic, spherical beads packed inside a column, typically made from materials like agarose, polyacrylamide, or dextran. These beads contain a network of pores with a defined range of sizes.
When a sample is introduced into the column, a liquid mobile phase carries the molecules through this bead-packed bed. Larger molecules are too big to enter the small pores within the beads, so they are excluded from the beads’ internal volume. These large molecules are restricted to the spaces between the beads and travel a more direct, shorter path through the column.
Smaller molecules, however, can readily enter and navigate through the pores and channels within the beads. This allows them to explore a larger volume and consequently take a more circuitous, longer path through the column. Because they spend more time diffusing in and out of the bead pores, their progress down the column is slowed.
As the mobile phase flows, the largest molecules, which bypass the beads, elute from the column first. Molecules of intermediate size enter some pores, taking a moderately longer path, and elute after the largest ones. The smallest molecules, which can access most, if not all, of the pores, are retained for the longest time and elute last. This creates a separation based on molecular size, with larger molecules exiting the column before smaller ones.
The Essential Parts of the System
A GFC system comprises several interconnected components. The chromatography column is a cylindrical tube packed with porous beads that serve as the stationary phase. These columns vary in size and material, and their packing is important for effective separation.
The mobile phase, typically an aqueous buffer solution, flows through the column. This buffer acts as the carrier for the sample molecules, moving them through the stationary phase. The choice of mobile phase depends on the molecules being separated, ensuring their stability and solubility throughout the process.
A pump delivers the mobile phase and sample through the column at a controlled and consistent flow rate. Maintaining a precise flow is important for reproducible separations and accurate elution times.
As molecules exit the column, a detector monitors their presence and concentration. Common detectors include UV-Visible detectors, which measure the absorption of ultraviolet light, often used for proteins that absorb at specific wavelengths like 280 nm. Other types include refractive index detectors. The detector generates a signal recorded as an elution profile, or chromatogram, indicating when different molecules elute from the column.
Why Gel Filtration Chromatography Matters
GFC is a valuable technique in various scientific and industrial settings due to its ability to separate molecules gently and effectively. One of its primary applications is in protein purification, where it helps isolate target proteins from complex mixtures based on their size. This is often used as a final polishing step after other purification methods.
The technique is also widely used for desalting, which involves removing small salt molecules from solutions containing larger biomolecules like proteins. Since salt ions are significantly smaller than proteins, they enter the bead pores and are retained, allowing the larger proteins to elute quickly and separately from the salts. This process, also known as buffer exchange, is important for preparing samples for subsequent experiments or storage.
Beyond purification and desalting, GFC is applied for determining the molecular weight of unknown molecules by comparing their elution volumes to those of known standards. It can also be used to assess the purity of a sample and to separate aggregates or degradation products from intact molecules. GFC is widely used in biochemistry, molecular biology, and biotechnology due to its versatility and mild operating conditions.