Size exclusion chromatography (SEC) separates components of a sample on the basis of their molecular size. Differential exclusion or inclusion of the molecules is achieved via filtration through a gel that contains spherical beads. These beads have pores of a specific size distribution so as to include or exclude molecules of different sizes when they pass through the gel.
SEC is also known by other names such as exclusion chromatography, gel filtration chromatography, liquid exclusion chromatography, and gel permeation chromatography.
The Principles of SEC
The separation of proteins and other compounds in SEC is achieved based on the size of the molecules in the sample. The technique uses a gel that contains porous spherical beads as the packing material in the chromatography column. Based on the size distribution of the beads, small molecules in the sample diffuse into the pores due to which their movement is slowed down. In contrast, the larger molecules do not pass through the pores, move more quickly through the beads, and are eluted. Thus, molecules are separated on the basis of their size by leaving the column in order of decreasing molecular weight.
Results obtained from this size-based separation of molecules allow a calibration curve to be constructed. This can be used to estimate the molecular weight of an unknown molecule in the sample by enabling comparison with proteins or polymers of known molecular weight.
Fractionation and desalting are the two common types of separations carried out using SEC.
In the desalting of proteins or nucleic acids, the exclusion limit of the gel is kept smaller than the size of the molecule of interest. Thus the molecule of interest is eluted out while smaller molecules are trapped in the pores of the gel.
In fractionation it is ensured that the molecules of interest fall within the gel’s fractionation range. Thus, analytes having different molecular weights are separated inside the gel matrix.
The Factors Affecting Resolution in SEC
The gel selection and operating conditions of SEC can vary depending on the specific application.
Increase in column length increases the resolution and increase in column diameter results in high bed volume and hence higher column capacity. The fractionation range and the exclusion limit can be controlled by varying pore size. The smaller the particle size of the gel, the higher the resolution achieved.
This needs to be moderate for the best resolution. This is because moderate flow rates give enough time to the molecules to make use of the entire surface area of the gel beads in the stationary phase, which allows smaller molecules to take time to enter the pores. This results in better separation of molecules of different sizes. Slow flowrates cause over diffusion of peaks and thus reduce the resolution.
Both overpacking and underpacking can be detrimental to the degree of resolution. Resolution is also drastically affected by the presence of dead volume on top of the column as the molecules in the sample diffuse before entering the column bed, which gives rise to broad bands or wider peaks.
Method of Detection
UV is the most widely used mode of detection and measurement of proteins during analysis of SEC. Aromatic amino acids like tryptophan respond better to near UV or longer wavelengths. However, higher wavelengths offer a higher linear dynamic range and lower wavelengths give better sensitivity and enables analysis of proteins present in low concentrations.