What is size exclusion chromatography?
Size exclusion chromatography (SEC), also known as gel filtration chromatography, is a chromatographic method that separates molecules based on their particle size (actually particle's Stokes radius). It is usually applied to large molecules or macromolecular complexes such as proteins and polymers.
What is the principle of size exclusion chromatography?
Typically, when samples enter into the column, different molecules will have different elution rates. The small particles trapped into the stationary phase pore system can go through a total volume (the sum of the entire pore volume and the inter-particle volume). They will elute for longer time. In the contrary, larger molecules simply pass by some stationary phase pores as they are too large to enter them. Therefore larger molecules elute through the column faster than smaller ones. That is to say, the smaller the molecule is, the longer the retention time will be.
Each size exclusion column has a range of molecular weight for separation. The exclusion limit for upper end of the column is that the molecules weight is too large to be trapped in the stationary phase. The lower end of the range is defined the molecular weight of the molecules which is small enough to enter all pores of the stationary phase. It should be noticed that particles of the same size or similar size may not separate from each other by SEC.
What is size exclusion chromatography used for?
This technique is widely used for the separation and purification of proteins. According to standard proteins, this technique is also useful for determination of molecular weight (Mw) and particle size. SEC is the official technique.
Chromatographic columns for SEC
| column | Mw range (Da) | sample | flow rate limit (ml/min) |
|---|---|---|---|
| Superdex Peptide 10/300 GL | 100-7,000 | peptide | 1.2 |
| Superdex 75 10/300 GL | 3,000-70,000 | polypeptide/protein | 1.5 |
| Superdex 200 10/300 GL3 | 10,000-600,000 | protein | 1 |
