Structural Biochemistry/Proteins/Purification/Gel-filtration chromatography

General Information

Gel-filtration chromatography is the simplest and mildest technique that separates molecules based on their size difference (also known as hydrodynamic volume). This way each polypeptide is purified from other different sized polypeptides. Gel-Filtration Chromatography is also known as Size-Exclusion chromatography, Molecular-Exclusion chromatography or Molecular Sieve chromatography.

When an organic solvent is used as a mobile phase, chemists tend to call it Gel permeation chromatography. This technique is used to analyze the molar mass distribution of organic-soluble polymers. It was invented by Grant Henry Lathe and Colin Ruthren who were working at Queen Charlotte's Hospital in London, United Kingdom.

The sample solution is applied to the top of a column containing a stationary phase of porous beads made of an insoluble but highly hydrated polymer such as dextran or agarose. These beads span a relatively narrow size range. When an aqueous solution of molecules of various sizes is passed through a column containing these 'molecular sieves,' small molecules can enter them but the large ones cannot. As a result, the big molecules are located in the solution around the beads while the smaller ones are able to penetrate the beads and the solution between them. The most important point is that large molecules flow more rapidly through this column and emerge first because a smaller volume is accessible to them and this is what enables the collection of fractions where the first set collected contains bigger molecules and the last set contains smaller molecules. And, theoretically, molecules that have the same size should elute simultaneously. An elution diagram, or a chromatogram, can be constructed to verify complete separation.

Within the size range of molecules that are subjected to gel-filtration chromatography and are separated by a particular pore size of beads in the column, there is a linear relationship between the relative elution volume of a substance (i.e., the volume of the fractions in which the molecule is found)and the logarithm of its molecular mass (this is assuming that the molecules have similar shapes). If a given gel filtration column is calibrated with several proteins of known molecular mass, the mass of an unknown protein can be estimated by its elution position.

Analogy

An analogy to understand (this is CONCEPTUAL, not even remotely a literal representation of what happens in ME chromatography) why gel filtration works is to picture several whiffle balls (or sponges or Swiss cheese-whatever cratered object works for you) suspended in a glass tank. Now imagine that you have a mixture of sand, small marbles, and golf balls in a bucket; you dump it in. As you watch, first the golf balls reach the floor of the tank, then the marbles, and finally a layer of sand settles. Why? Essentially all of the sand goes into the holes of the whiffle balls(or Swiss cheeses or sponges), and it tends to fall from the interior of one whiffle ball to the interior of another, significantly slowing passage of the sand to the bottom of the tank. The marbles are only slightly smaller than the holes in the whiffle ball, so they sometimes fall into the holes on the way down but also sometimes bounce off; again, the whiffle balls slow their progress, but to a lesser extent. The golf balls are way too big to fit the holes of a whiffle ball, and so they push straight through the whiffle balls--the fastest and most direct route. Key: sand=small molecules; marbles=medium molecules; golf balls=large molecules; whiffle balls=porous beads; tank of water=column & aqueous solution

General Procedure

First, apply the sample or mixture to the top of the gel filtration column that is filled with insoluble carbohydrate polymer beads with different sizes of pores. [Agarose, dextran, or polyacrylamide are usually used as the stationary phase (gel medium) under low pressure. However, silica or cross-linked polystyrene can also be used as a stationary phase under a higher pressure.] As the mixture flows towards to the bottom of the column, large molecules will emerge first because they cannot distribute inside the beads - they can only exist in between the beads in the column and they have a smaller accessible volume, whereas smaller molecules can distributed into both inside and between the beads. Thus, small molecules will take a long time to elute because they have a longer tortuous path before they come to the exit of the column.

Gel-Filtration Chromatography is commonly used for analysis of synthetic and biological polymers such as nucleic acid, proteins, and polysaccharides. A downfall to this technique is that the stationary phase may also interact in an undesirable way with a molecule and affect its retention time.

Gel-Filtration Chromatography should not be confused with gel electrophoresis, where electricity is applied to create an electric field to separate molecules through the gel towards the electrode (anode and cathode) depending on their electric charge.