# Size Exclusion Chromatography

What can you infer from the term ‘’size exclusion’’? Some molecules excluded on the basis of size? The exclusion of molecules on the basis of size lays the foundation for this very interesting addition in our chromatographic series called size or molecular exclusion chromatography. Other names for size/molecular exclusion chromatography are molecular sieve chromatography and molecular size chromatography. Size exclusion chromatography can be further classified on the basis of the type of stationary phase and mobile phases used.

In case, you are still unfamiliar with chromatographic stationary phase and mobile phases, we recommend: difference between stationary phase and mobile phase in chromatography. You may also like to read first about what is chromatography and come back here to continue with us on size exclusion chromatography.

## What is size exclusion chromatography

Size exclusion chromatography is a column chromatographic technique in which separation occurs on the basis of size and shape i.e., the hydrodynamic dimensions of the analyte molecules. It was first developed in 1955. The stationary phase in this case is a porous gel while the mobile phase solvent sweeps pass it under the action of gravity. No external force, temperature or pressure conditions are required for size exclusion chromatography. Size exclusion chromatography is particularly suitable for separation of bulky components and macromolecules such as polymers, proteins, nucleic acid and/or polysaccharides etc.

## What is stationary phase in size exclusion chromatography

Porous gels such as silica gel is the most popular stationary phase choice for performing size exclusion chromatography. Other options include dextrin, agarose and polyacrylamide etc. The stationary phase is packed into a column. Highly crosslinked gels are composed of extremely fine pore sizes.  Finer the pore size of the gel, better the chromatographic separation thus a well resolved chromatogram. No chemical force of attraction is involved in analyte retention onto the stationary phase. Rather, the analyte molecules smaller than the stationary phase pore size, gets trapped and retained in it.

## What is mobile phase in size exclusion chromatography

The mobile phase in size exclusion chromatography are the solvents that transport the analyte mixture down the stationary phase packed column. The mobile phase solvents could be inorganic, aqueous-based such as a neutral phosphate buffer. Similarly, non-aqueous and organic solvents such as pyridine and tetrahydrofuran can also be incorporated.

## Working principle of size exclusion chromatography

Smaller molecules from the analyte mixture, having a smaller size than the pore size of the stationary phase gel, gets trapped. On the other hand, molecules larger than the pore size are excluded i.e., they easily pass through the gel and get eluted out of the column with the mobile phase solvent. Thus, on the basis of molecular size, the components elute out of the column at varying time intervals and give distinct chromatographic peaks.

## How to perform size exclusion chromatography

The following step-by-step guide explains a size exclusion chromatographic process.

Step I: Stationary phase pre-treatment

The stationary phase material is first soaked overnight into the solvent to be used as the mobile phase.  This allows the stationary phase material to take up the solvent and turn into soft, flexible gel beads. The internal diameter of the beads differs as well as differently sized pores are formed inside these beads. This pre-treatment is carried out in order to avoid any risk of stationary phase cracking into the column in case gel expansion occurs while performing chromatographic separation.

Step II: Column packing

The bottom end of a glass chromatographic column is blocked with a cotton plug followed by a thin layer of sand. The porous beads are then packed into this column. It is allowed some time to settle down under gravitational action and form a close packing afterwards.

A sample slurry is prepared by adding the mobile phase into the analyte mixture. This mixture is consequently loaded onto the stationary phase packed column. Another layer of mobile phase is then introduced on top of it and allowed to travel down the column, carrying the analyte components with it.

Step IV: Analyte separation

The molecules bigger than the pore size such as 4 shown in the figure below gets eluted out of column readily. Smaller molecules such as 1,2 and 3 gets trapped into the pores. 3 will be the second to elute out of the column followed by 2 while 1 (the smallest molecule) will stay trapped for long thus eluting out of the column at last. In this way, molecules 1, 2, 3 and 4 gets separated out of a complex sample mixture. A signal is consequently sent to the detector which generates a chromatographic response.

For detectors, refer to the detectors used for high-performance liquid chromatography (HPLC).

Analyte separation by size exclusion chromatography can be mathematically interpreted using the elution equation, as discussed in the next section.

### The Elution Equation

The elution equation is a numerical interpretation for the total mobile phase volume required to elute an analyte component out of the column. The interstitial spaces present between stationary phase particles in a non-uniformly packed column (as that used for size exclusion chromatography) are called ‘voids’’. We first need to learn some basic terms before we get introduced to the elution equation.

The elution equation for size exclusion chromatography can then be written as equation 1 below:

Vt= Vo + Vg + Vi   Equation 1

Vo depends on a series of factors which can cumulatively be expressed as equation 2

Vo = Ve-kd (Vi) Equation 2

where kd represents a distribution coefficient inside the chromatographic column. The equation 2 can be rearranged as equation 3 to make kd the subject of the formula.

$kd=\frac{Ve-Vo}{Vi}$                         Equation 3

For very large molecules incapable of penetrating the pores of the stationary phase, Ve=Vo.

Thus equation 3 gives kd =0. On the other end, for very small molecules that readily penetrate and get trapped into the stationary phase for long, kd= 1. Henceforth, a kd value between 0 to 1 facilitates a good chromatographic separation by size exclusion chromatography.

## Types of size exclusion chromatography

There are two further types of size exclusion chromatography. The distinction is made on the basis of the types of stationary phase, mobile phase and the purpose of analysis, as you can see in the table below.

## Importance of size exclusion chromatography

As mentioned earlier, size exclusion chromatography plays a significant role in chromatographic separation and purification in a large bulk. It can be used for both qualitative as well as quantitative analysis.

Some of the most useful applications of size exclusion chromatography are:

• Molecular weight determination for polymers: A series of standard compounds passed through a size exclusion chromatographic column, can help obtain a calibration curve. The calibration curve is plotted by using log of molecular weight for the standards versus the volume of mobile phase required for their elution from the column (Ve). This calibration curve can help determine the unknown molecular weight of polymers, macromolecules etc., once their Ve is known. However, this method for molar mass analysis is not very reproducible/reliable.
• Protein complex association: Pure protein molecules and a protein complexing ligand are simultaneously passed through a size exclusion stationary phase. Depending upon the difference in the sizes of the pure molecules and those that got complexed with the ligands, two different chromatographic peaks will be obtained. The difference between the two peaks can help us in determining the amount of protein that binds with the ligand.
• Protein integrity: A denaturing agent can be passed through a size exclusion column along with the protein sample. Denaturation unwinds the globular structure of the protein molecules. Compactly packed versus denatured protein molecules elute out of the column at differing rates, requiring different elution volumes. In this way, the level of folding in the targeted protein molecules can be adequately determined.
• Protein desalting: Unwanted, small molecules can be removed from a targeted sample by size exclusion chromatography. The small molecules such as salt get trapped into the porous gels, while targeted analyte components (proteins) get eluted out of the column.

For more in-depth information on size exclusion chromatography (SEC) and its applications in polymer chemistry you may like:  SEC for nanoparticulate drug delivery systems.

Conclusion:

Size exclusion chromatography is an efficient and a fast chromatographic tool that aids analysis especially in polymer science. But low resolution and limited reproducibility are the two major concerns associated with its use.

References:

1. Mori, S. and H. G. Barth (1999). Size Exclusion Chromatography Springer.
2. Nagy, K. and K. VÉKey (2008). Chapter 5 – Separation methods. Medical Applications of Mass Spectrometry. K. Vékey, A. Telekes and A. Vertes. Amsterdam, Elsevier: 61-92.