Gel Filtration Chromatography (GFC) and Size Exclusion Chromatography (SEC) describe the identical separation method, which sorts molecules based purely on their physical size in solution. SEC is the overarching, modern term used universally to describe the technique. The difference between them is primarily one of historical context and the type of sample being analyzed. GFC specifically refers to the application of this method when separating biological macromolecules, such as proteins or nucleic acids, using an aqueous buffer as the liquid phase.
How Size Exclusion Separates Molecules
The separation mechanism in size exclusion chromatography relies on a stationary phase packed with porous beads, which act like a molecular sieve. These beads contain pores of a precisely controlled size distribution, determining the range of molecules that can be separated. The mobile phase, or solvent, carries the sample through the column and around the porous particles.
Separation is based on the molecule’s hydrodynamic volume, which is the effective size the molecule occupies when dissolved in a solution. Molecules larger than the largest pores are completely excluded and cannot enter the porous matrix. These large molecules flow around the beads, taking the shortest path, and therefore elute from the column first.
Smaller molecules can penetrate the pores to varying degrees, depending on their size relative to the pore dimensions. The smallest molecules can enter the full volume of the pores, significantly increasing their path length through the column. Consequently, these smaller molecules are retained longer and elute last, emerging in order of decreasing hydrodynamic volume. This process is a non-interactive separation, meaning molecules do not chemically bind to the stationary phase.
Clarifying the Terminology Difference
Gel Filtration Chromatography is the older, traditional name for this technique, originally coined for separations involving water-soluble biomolecules. The “gel” refers to the early stationary phases, which were often soft, porous materials like cross-linked dextran or polyacrylamide gels. This term remains widely used in biochemistry laboratories focusing on protein purification and analysis.
Size Exclusion Chromatography (SEC) is the term preferred by international scientific organizations, used regardless of the mobile phase or sample type. When the separation is performed on synthetic polymers using organic solvents, the technique is often called Gel Permeation Chromatography (GPC). GPC, GFC, and SEC all share the identical physical principle of separation based on hydrodynamic volume. GFC is accurately viewed as a specific category of SEC, differentiated only by its aqueous mobile phase and biological application.
Key Applications and Method Constraints
Size exclusion chromatography is widely used for practical tasks in both research and industrial settings, particularly for biopharmaceuticals. A major application is desalting or buffer exchange, where the target macromolecule is rapidly separated from much smaller salt ions or reaction byproducts. It also serves to estimate the molecular weight of an unknown molecule by comparing its elution time to known standards. SEC is frequently used to analyze protein samples for the presence of aggregates, which are larger clumps of the target molecule.
The technique’s reliance purely on size also imposes certain limitations. SEC offers lower resolution compared to other chromatographic methods, such as ion-exchange or affinity chromatography, especially when separating molecules of very similar size. Separation is only effective within a specific molecular weight range defined by the pore size of the column matrix. The method does not separate based on charge, binding affinity, or hydrophobicity, meaning molecules with the same hydrodynamic volume will co-elute.