Size-exclusion chromatography

Size exclusion chromatography (SEC) is a chromatography method that separates molecules according to their sizes and in some cases molecular weight. It is also known as gel filtration chromatography, gel permeation chromatography, and molecular sieve chromatography. LT Biotech offers a product line for SEC chromatography.

As a rule, the smaller molecules will move slower in this technique. To do so, the technique uses a chromatography resin made of spherical beads with pores of a specific size range. When a liquid media is applied to the size exclusion chromatography resin, smaller molecules will enter and diffuse into the beads’ pores, slowing them down. Meanwhile, larger molecules will be excluded from entering the bead’s pore, resulting in a quicker flow through the size exclusion chromatography column. The maximum size of the molecules able to enter the pore is referred to as the exclusion limit.

The steps to perform purification with size-exclusion techniques are as follow:

1. The column is flushed with the mobile phase.
2. The sample is injected into the circuit of the mobile phase, going into the column and interacting with the size exclusion resin.
3. The exit of the column collects the fractions. This is where a detector is often added, to distinguish the different fractions, which are then collected.

Size exclusion chromatography will often be coupled to an analytical method to detect the presence of molecules of interest in the eluted liquid.

The options for analysis can be very diverse, depending on the detection needs and the molecules targeted, for example:

• Ultraviolet (UV) absorption
• Fluorescence
• Refractive index (RI)
• Infrared detectors
• Multi-angle laser light scattering (MALLS)
• Charged aerosol detection (CAD)
• Density detectors
• Small-angle X-ray scattering (SAXS) detectors

Common column materials are silica, polyacrylamide, dextran, and agarose. Many different solvents can be used, from non-polar to aqueous, for example, tetrahydrofuran (THF) is a popular non-polar solvent.

Size exclusion chromatography can be used for a variety of applications

A common application is “desalting”. The molecule of interest, for example one or multiple proteins or nucleic acids, will flow through the column due to its large size. But smaller molecules and salts will be retained longer by the size exclusion chromatography resin, making them flow slower. This way, the initial eluant contains the product of interest, with almost all salts still stuck in the size exclusion gel.

Another application is fractionation, where molecules of varying molecular weights are separated by the size exclusion resin and can be separated in multiple fractions.

As a general rule, desalting requires an exclusion limit much smaller than the molecule of interest.

Because desalting has such a large difference between molecule sizes, the sample volume can be a larger part of the total volume (up to 30%), and larger & wider columns can be used.

To the contrary, fractionation requires that the weight of the molecule of interest is within the fractionation range of the gel, as fully excluded molecules (too big ones) will not be slowed down at all and will all gather in the first fraction of the elution.

Regarding fractionation of a very wide range of molecular weights mixed together, a series of several columns connected in series, each with different pore sizes, can be used. It will properly split the initial sample by all the possible molecular weight fractions. This is sometimes referred to as mixed gel, linear size-exclusion chromatography, or multi-pore gel.

A significant advantage of size exclusion compared to other forms of chromatography is that the molecules do not bind to the column’s matrix, reducing the effect of the buffer’s composition, especially compared to chromatography techniques like ion-exchange chromatography. This results in much more diverse possible conditions for the sample, which can be varied according to the source of the product, as well as modified to fit further purification steps, analysis equipment, or storage conditions. There is also normally no sample loss, as the product does not chemically interact with the column’s matrix.

A limitation of size exclusion chromatography is the limited resolution of the technique, with different molecular species needing most of the time more than a 10% difference in molecular weight to be distinguished and separated.

It will also not separate according to chemical properties or nature, and depending on the initial sample, will mix together protein or peptides with nucleic acid for example. It is also a low-resolution method when it comes to estimating the molecular weight of a purified product, with other techniques required for an exact measurement.

Another risk to control for is residual silanol interactions, mostly between larger bio-macromolecules and a silica-based stationary phase. Coating of the silica beads, for example with dextran, can help reduce this issue.

SEC resins can be supplied in 25 ml, 100 ml, 500 ml, 1L, 5 L, 10 L and 20 L plastic bottles or tins, and can be prepacked into 1 ml, 4.7 ml and 5 ml columns, suitable for the ÄKTA™ system. The catalogue number consists of eight digits, in the format XXX-ZZZZZ. XXX is the product code and ZZZZZ is the pack size in ml. For example, 701-00025 is a 25-ml bottle of Persefose 4FF, and 200-10000 is a 10 L tin of Persecryl 200. For detailed information on pack types and quantities please contact your local distributor or refer to the product list.