Gel Filtration Chromatography

Gel filtration chromatography is a separation based on size. It is also called molecular exclusion or gel permeation chromatography. In gel filtration chromatography, the stationary phase consists of porous beads with a well-defined range of pore sizes. The stationary phase for gel filtration is said to have a fractionation range, meaning that molecules within that molecular weight range can be separated.

Proteins that are small enough can fit inside all the pores in the beads and are said to be included. These small proteins have access to the mobile phase inside the beads as well as the mobile phase between beads and elute last in a gel filtration separation.

Proteins that are too large to fit inside any of the pores are said to be excluded. They have access only to the mobile phase between the beads and, therefore, elute first.

Proteins of intermediate size are partially included - meaning they can fit inside some but not all of the pores in the beads. These proteins will then elute between the large ("excluded") and small ("totally included") proteins.

Consider the separation of a mixture of glutamate dehydrogenase (molecular weight 290,000), lactate dehydrogenase (molecular weight 140,000), serum albumin (MW 67,000), ovalbumin (MW 43,000), and cytochrome c (MW 12,400) on a gel filtration column packed with Bio-Gel P-150 (fractionation range 15,000 - 150,000). When the protein mixture is applied to the column, glutamate dehydrogenase would elute first because it is above the upper fractionation limit. Therefore it is totally excluded from the inside of the porous stationary phase and would elute with the void volume (V0). Cytochrome c is below the lower fractionation limit and would be completely included, eluting last. The other proteins would be partially included and elute in order of decreasing molecular weight.

These separations can be described by this equation

where Vr is the retention volume of the protein, V0 is the volume of mobile phase between the beads of the stationary phase inside the column (sometimes called the void volume), Vi is the volume of mobile phase inside the porous beads (also called the included volume) and K is the partition coefficient (the extent to which the protein can penetrate the pores in the stationary phase, with values ranging between 0 and 1).

In the mixture of proteins listed above, the partition coefficient (K) for glutamate dehydrogenase would be 0 (totally excluded), K = 1 for cytochrome c (totally included) and K would be between 0 and 1 for the other proteins, which are within the fractionation range for the column.

In practice, gel filtration can be used to separate proteins by molecular weight at any point in a purification of a protein. It can also be used for buffer exchange - a protein dissolved in a sodium acetate buffer, pH 4.8, can be applied to a gel filtration column that has been equilibrated with tris buffer, pH 8.0. Using the tris buffer, pH 8.0, as the mobile phase, the protein moves into the tris mobile phase as it travels down the column, while the much smaller sodium acetate buffer molecules are totally included in the porous beads and travels much more slowly than the protein.