Structural Biochemistry/Proteins/Purification/Gel-Filtration chromatography

Gel-filtration chromatography, also known as 'size exclusion chromatography', 'molecular exclusion chromatography' or 'molecular sieve chromatography' is the simplest and mildest technique that separates molecules based on their size difference (hydrodynamic volume). This approach allows each polypeptide to be purified from other different sized polypeptides by passing through a gel filtration medium packed into the column. Unlike ion-exchange or affinity chromatography, fractions passing through the column do not bind to the chromatography medium. The big advantage of Gel-filtration chromatography is that the medium can be varied to suit the properties of a sample for further purifications.

A Gel Filtration column

When an organic solvent is used as a mobile phase, chemists tend to call it Gel permeation chromatography. The buffer or organic solvents used as the mobile phase are chosen based on the chemical and physical properties of the specific protein sample. The stationary phase of the column is simply the carbohydrate polymeric beads and the mobile phase goes through the stationary phase at a different speed depending upon the size of the molecule. 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.

Gel-filtration chromatography can be applied in two different ways: for group separations and high resolution fractionation of biomolecules. The group separation technique separates compounds in a sample into groups based on the size range. This technique is used for purification of a sample from high or low weight contaminants. The high resolution fractionation of biomolecules is a more precise technique. It can be used for isolation of one or more components in a sample, separation of monomers from aggregates, to determine molecular weight, or to perform molecular weight distribution analysis. Gel-filtration chromatography is very suitable for biomolecules which are very sensitive to pH changes, concentration of metal ions, or co-factors.

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

The gel medium packed into the column is a porous matrix that consists of spherical beads, which have stable physical and chemical properties such as non-reactivity and lack of adsorption. The small molecules can enter the beads but the larger one cannot. The small molecules are distributed in the aqueous solution both inside and between the beads where as the large molecules are located in the solution between the beads. These beads are not soluble and are normally made from highly hydrated polymers such as dextran, agarose, or polyacrylamide. For commercial purposes, Sephadex, Sepharose, and Biogel are used. These commercial beads are about 100 miciro-meters in diameter and are used to separate proteins based on sized. Also, silica or cross-linked polystyrene can also be used as material for the beads under higher pressures. The pores and space between the particles is filled with a liquid buffer, which fills the entire column. The liquid filling the pore space is called a stationary phase and the liquid in the space between particles is a called mobile phase. Once the sample has been applied to the top of the column, it passes through the column along with the mobile phase from the top of the column to the bottom. Smaller molecules are able to cross and go through these polymer beads but large ones are not able to. Therefore, small molecules in the column are both inside the polymer beads and between them, whereas large molecules can only travel between the polymer beads. Since less traveling space is allowed for the larger beads, they tend to move faster down the column and they emerge first at the end of the column. Think of it this way. The molecules traveling down the column represent a faucet. If the faucet has a smaller volume of space to allow the water to travel, the water will come out faster and with greater force. The same concept applies here as well. Since less volume is accessible to the bigger molecules, they move much faster through the column than smaller molecules do. So, since the small molecules are stuck inside the beads, they tend to move slower. Theoretically, molecules that have the same size should elute simultaneously. An elution diagram, or a chromatogram, can be constructed to verify complete separation. Before separation of unknown sample, solutions with known biomolecules can be run in order to make a calibration curve, which later can be used as a reference for identifying of unknown molecules.

Utilization

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. A major drawback to this method is its difficulty in producing a high-resolution image. An alternative to this may be Discontinuous Electrophoresis. Disc electrophoresis uses gels with different pHs and the proteins produce sharp bands when they go from one gel to the other, which creates high-resolution images. This technique requires three different gels: the sample gel, the stacking gel, and the running gel. The proteins moves through the stacking gel and between the sample and running gels before the proteins enter them. This compresses the proteins and increases the resolution.

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. Besides, large molecules in Gel-filtration Chromatography migrate down the column first whereas small molecules in gel electrophoresis migrate down the gel first.

References

Viadiu, Hector. Biochemistry 114A Lecture. "Protein Techniques." 10/15/12