Size Exclusion Chromatography (SEC) is a technique used to separate components in a mixture based on their physical dimensions, or hydrodynamic volume. It is also known as Gel Filtration Chromatography (GFC) for biomolecules in aqueous solvents, or Gel Permeation Chromatography (GPC) for polymers in organic solvents. SEC utilizes a column packed with porous material to achieve separation.
The Fundamental Principle: Size Dictates Elution Order
The largest molecules exit the column first in Size Exclusion Chromatography. This separation is based purely on the physical size of the molecules, or their hydrodynamic volume, unlike methods relying on chemical interactions or charge.
The largest molecules travel the shortest path because they are unable to enter the microscopic pores of the packing material and are completely excluded from the internal volume of the beads. They flow only through the space surrounding the beads, which is the most direct route, ensuring they are the first to be eluted.
Understanding the Stationary Phase and Separation Mechanism
The stationary phase in SEC is composed of spherical, porous beads packed tightly into a column. These beads are typically made from cross-linked polymers such as dextran, agarose, or polyacrylamide for aqueous separations. The beads act as a molecular sieve, possessing a defined distribution of pore sizes that governs the separation range of the technique.
The physical mechanism relies on the differential access molecules have to the internal pore volume. Molecules significantly larger than the largest pores are unable to enter the beads. This defines the column’s Exclusion Limit, the size threshold above which all molecules are completely excluded from the pores and follow the shortest path.
Exclusion and Permeation
Molecules that fall below this exclusion limit are separated based on their size relative to the pore network. Intermediate-sized molecules partially penetrate some pores, taking a longer, more circuitous path. The smaller the molecule, the more internal pore volume it can access, causing it to be temporarily retained longer.
Molecules smaller than the smallest pores are fully permeated because they access the entire available pore volume, traveling the longest path and eluting last. This difference in travel time results in an elution order where size is inversely proportional to elution time.
Key Parameters for Analyzing SEC Results
Interpreting the results from a size exclusion chromatogram requires understanding a few specific volume measurements. The Elution Volume (\(V_e\)) is the total volume of mobile phase required to wash a specific substance out of the column and into the detector. This \(V_e\) is the primary measurement used to determine a molecule’s size in SEC.
Two other volumes define the boundaries of the separation window. The Void Volume (\(V_o\)) is the volume of mobile phase located in the spaces between the porous beads. This is the volume where all molecules larger than the exclusion limit travel, meaning the largest molecules will always elute at \(V_o\).
The Total Volume (\(V_t\)) is the sum of the void volume and the entire internal pore volume within all the beads. The elution volume (\(V_e\)) for any molecule must always fall between \(V_o\) and \(V_t\). Molecules that elute at \(V_o\) are too large to be separated, while molecules that elute near \(V_t\) are too small to be separated effectively. The Exclusion Limit sets the upper limit for effective separation.
Common Applications of Size Exclusion Chromatography
Size Exclusion Chromatography is a versatile technique used widely across biochemistry and polymer science.
Common applications include:
- Separation of very large molecules from very small molecules, known as desalting or buffer exchange, which rapidly removes small salts or unincorporated reagents.
- Purification of macromolecules, such as proteins, antibodies, and synthetic polymers.
- Separating aggregates (e.g., protein dimers or trimers) from the desired single-unit (monomer) protein.
- Estimating molecular weight and determining the molecular weight distribution of unknown samples by calibrating the column with known sizes.