Size exclusion chromatography
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Size exclusion chromatography (SEC) is a chromatographic method in which molecules are separated based on their size. SEC is also known as gel permeation chromatography, or gel filtration chromatography, but these refer specifically to instances where the chromatographic medium is a gel.
SEC is a widely used technique for the purification and analysis of synthetic and biological polymers. Biologist and biochemists typically use SEC in the gel mode, under low pressure and with polymeric gels (often on sugar polymer supports). Polymer chemists typically use SEC in high pressure mode (typically with silica or crosslinked polystyrene supports.
Theory
The theory behind size exclusion chromatography is simple, although to individuals experienced in other chromatographic techniques, the result can be counterituitive at a first glance. In SEC, the injection medium appears after the separated material, which is not the case in most other chromatographic techniques, and the size:velocity relationship is reversed relative to electrophoresis, another chromatographic technique which separates based on molecular size.
SEC operates by having a stationary phase consisting of a porous material. Typically these are beads with crevasses or even channels. For any given molecule there is a probability of being trapped in the bead. This probability scales downward with size. Molecules which are too big to fit into these beads must go around them completely. Therefore larger molecules travel through the column faster.
A Simplified Example
Let's say we have two types of particle we would like to separate, a large and a small particle. Our beads (which completey exclude the large particle) occupy 80% of the unit volume, and 50% of the beads (40% of the total volume overall) are a channel accessible to just the small particle. 25% of the beads (20% overall) are inaccessible to the small particle but accessible to solvent, and 25% is solid. Our column contains 100mL of total volume (beads + solvent), and we flow liquid through the column at .8 mL/min.
Note that the total volume accessible to solvent is 80 mL, so it will take 100 minutes for the solvent to flow through. The small particles can access 60 mL of space, and they will elute in 75 minutes. The big particles can only see 20 mL of space, and will elute from the column in 25 minutes. Since the big particles are completely excluded from the beads, their elution volume (20mL) is known as the void volume, and the solvent volume is known as the column volume.
Caveats to the simplified example
Note that in true situations, particles do not typically have an exact amount of space in which they can reside, and their probability of falling into any given bead's accessible volume is stochastic. Therefore, elution curves resemble gaussian distributions. Other factors, such as non-specific adsorption, can influence retention times, although great care is taken by column manufacturers to use stationary phases which are inert and minimize this issue. Like other forms of chromatography, Increasing column length tightens the resolution, and increasing the column diameter increases the column's capacity Proper column packing is also important to maximize resolution: an overpacked column can collapse the pores in the beads, resulting in loss of resolution; conversely, and underpacked column can reduce the relative volume accessible to smaller species (by increasing the volume that is accessible to all species)
Finally, unlike ion-exchange chromatography or affinity chromatography, size-exclusion chromatography relies entirely on diffusion, so samples necessarily undergo a dilution, and resolution is adversely affected by increasing the injection volume. Slower solvent speeds will improve resolution to a certain point, but eventually nonspecific diffusion overtakes as a confounding factor.ar:تفريق لوني بعبور الهلام cs:Gelová chromatografie fr:Chromatographie d'exclusion stérique sv:Gelfiltrering