Size exclusion chromatography a solute molecule relies on sorting of molecules of different hydrodynamic radius based on the time these molecules spend within the matrix. In this mode of chromatography porous beads made of a neutral support are used to produce a long column. The beads are riddled with tunnels of varying size. Molecules running through this type of column have to solve a maze which becomes more complex the smaller the molecule is as the small molecules have more potential tunnels they can access. Thus, partitioning occurs as a result of the molecules spending more or less time within the volume of the beads which make up the column. Molecules with large hydrodynamic radius elute early on in the gradient; molecules with smaller radii elute later.

Chemistry:

Although the support chemistry used for SEC matrices is chosen to reduce non-specific interactions, partial charges on the matrix can generate mixed-mode chromatography. Many people use an ionic component in the buffer to shield these charges. Additionally, hydrophobic proteins may bind to the matrix and chose not to elute. It is wise therefore to determine the extent of recovery of your sample from a SEC matrix prior to loading the entire material.

Pore Size:

Resins are usually classified by manufacturers based on their capacity to separate different sizes of a hypothetical, globular protein. The lower range is the range below which all molecules will see the entire internal volume of the beads allowing for no selection below this size. The upper range is the range at which molecules are completely excluded from seeing the inside of a bead also allowing for no separation. There is a linear range between these two extremes at which decent separation of molecules occurs. This range is what is usually reported for each matrix. The pore size used is dependent on the range of separation desired. Smaller pores are used for rapid desalting procedures or for peptide purification. Larger ones are used for small proteins, while very large ones are used for biological complexes. Complexes with an equivalent hydrodynamic radius of a 1,000 MDa globular protein can be separated using this type of resin.

Pouring a column:

The resolution of SEC columns is notoriously dependent on good pouring technique and maintenance. These columns need to be re-poured if they dry out! (Good technique is to never let any column run completely dry) To pour this column, degas your buffer, and allow your matrix, if supplied as a dry powder, to swell (min. overnight). Matrices can swell dramatically, so be sure to check how much is needed to fill your column! Attach a funnel or pouring device to the top of your column and pour a dilute slurry of your matrix into the top until the attached funnel is full. Allow for slow settling of the beads. One can siphon off the buffer on top after the resin has settled and re-pour more slurry into the top as needed. Be sure not to leave much buffer space at the top of the column as diffusion can occur in this space at the cost of resolution. If loading manually, weigh down your sample by adding glycerol. This will allow for stacking of the sample onto the top of the column.

Applications:

SEC is not a high resolution technique. As a rule of thumb, do not attempt to separate two components with this technique unless they differ by at least two-fold in molecular weight. This type of chromatography can be used to remove small molecule contaminants from protein preps., is popular as a polishing steps for protein manufacture, and is used to determine the solution subunit composition of a multimeric protein, and to isolate different multimers from each other.

SEC is usually run without a gradient using a single buffer at a slow, constant flow rate.

Buffer 1: 30 mM Tris, 150 mM NaCl

Buffer 2: Phosphate Buffered Saline

Buffer 3: 30 mM Tris, 150 mM NaCl, 8M urea (SEC can be run in denaturants, but beware of the dramatic change in hydrodynamic radius as your molecule now deviates significantly from the theoretical globular protein)