Size-exclusion chromatography is a technique that has been around for more than half a century. As the name suggests, it allows separation of proteins and other molecules based on their size. The principle is quite simple; a chromatography medium containing pores is packed into a column; smaller molecules can penetrate more deeply into the pores than larger molecules. Consequently, larger molecules move faster through the column causing the molecules to be separated.
Fig. 1 SEC separation utilises the molecules own size differences
The selectivity of a SEC medium describes how well molecules of different sizes are separated and defines the medium's fractionation range. This range is controlled by the pore size distribution. Today’s SEC media cover a molecular weight range from 100 to 100 000 000 Da, from peptides to very large proteins and protein complexes.
SEC media can be made out of several different materials. For high-resolution protein separations, dextran, agarose, silica, hydrophilized vinyl polymers such as polyacrylamide or a combination of these polymers are commonly used. A common feature with these materials is low secondary interactions with proteins. Ideally, there should be no interaction at all but in reality no material is completely adsorption free.
Another important property of a SEC medium is the compatibility with common solvents and varying pH’s. Here, the materials differ. While the pH stability range of agarose, dextran and polyacrylamide is wide, silica dissolve at high pH and should not be used above a pH of 7.5. This makes it difficult to clean the medium properly and hence shortens the lifetime. Basic buffers cannot be used. An advantage, however, of silica-based matrices, is that they are available with smaller bead diameters than agarose-based matrices traditionally are. Importantly a small bead size increases the resolution of the separation.
Increase: A Novel Size Exclusion Chromatography Media Platform
Recently, a new generation of agarose-based SEC platform was launched allowing high-resolution analytical separations comparable to using silica-based resins. This is due to the beads of the media being smaller than the traditional agarose-based SEC media (average 8.6 µm) and having smaller particle size distribution. The media are based on a high-flow base matrix with good pressure/flow properties allowing high flow rates and thus shorter purification times and alkali resistant allowing rigorous cleaning leading to better reuse with minimal carry over and prolonged column lifetimes.
The two different media sizes have with different separation ranges
Superdex 200 Increase and Superose 6 Increase have different fractionation ranges and hence separate different types of proteins. This is illustraded by the chromatograms in fig 2. As a rule of thumb, Superdex 200 Increase is recommended for proteins below ~440 kDa and Superose 6 Increase is recommended for proteins and protein complexes above this size.
Figure2. Chromatograms showing high-resolution SEC of six standard proteins on (A) Superose 6 Increase 10/300 and (B) Superdex 200 Increase 10/300 GL.
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