When you have mastered this topic, you will
- understand the basic principles of gel filtration chromatography;
- know the terminology used in gel filtration chromatography.
Gel filtration chromatography operating principles
Gel filtration chromatography (GF)(also known as molecular exclusion chromatography) relies on a stationary phase consisting of spherical gel particles whose size and porosity are carefully controlled during manufacture.
When the mobile phase is passed through a column of this material, molecules are fractionated on the basis of their sizes and shapes. Small molecules (shown in blue in the diagram on the left) are able to diffuse into the pores, while larger molecules (shown in red) are excluded. This occurs repeatedly as the mobile phase moves down the column, and as a result, during the elution process, the small molecules are retarded with respect to the larger molecules. This causes the smaller molecules to appear in the later fractions of the eluate.
Size is not the only factor that influences the rate at which solutes are retarded. For a given pore size, rod-like molecules will tend to be excluded with respect to spherical molecules of the same size. In addition, certain molecules may have some affinity for the stationary phase as a result of non-covalent interactions.
Advantages of gel giltration chromatography
- Separation process is carried out under very mild conditions, from 37 ºC. to cold room conditions.
- High resolution can be achieved.
- A wide range of buffer systems may be used (buffer molecules and ions are generally excluded).
- Small molecules or ions that stabilize the biomolecules may be added to the buffer.
- Separation process is independent of ionic strength, so elution is carried out isocratically.
The ionic strength, Iof a solution is defined as I = ½Σcizi2, where ciis the molarity of the ithion, and zi its charge, the summation covering all the ions in the solution.An isocratic elution is one that takes place with a buffer of constant ionic strength.
- Gel filtration can be used for desalting and/or buffer exchange.
- Gel filtration can be used to obtain an estimate of molecular size.
- A wide range of porous gels are available commercially.
Parameters used in gel filtration chromatography
For a column with diameter, d and a bed of height, h, the bed volume or total volume,
Vt = π(d/2)2h
- The internal volume, Vi, the volume inside the pores of the gel particles.
- Vg, the volume of the gel that is impenetrable to the solvent.
- The void volume, V0, the volume of liquid between and outside the gel particles.
Therefore, Vt = Vi + Vg + V0
The elution volume, Ve, of a solute is the volume of eluant which leaves the column from the moment that the sample penetrates the gel bed to the moment that solute first emerges from the column.
The void volume, V0, is readily determined by measuring the elution volume of a macromolecule that is completely excluded. For this purpose, one frequently uses the polymer Blue Dextran 2000, with Mr ~ 2×106, whose progress through the column can be seen due to its blue colour.
The solvent regain of the gel is the mass of solvent (normally water) absorbed by the gel per gram of dry gel. It is obtained by weighing a small quantity of dry gel, suspending it in water until it has fully swelled, filtering the gel off, and weighing it. This enables us to calculate Vi, which then equals xy/ρ, where x is the mass of dry gel in the column, y, the solvent regain, and ρ, the density of the solvent.
The partition coefficient, Kav for a solute is given by:
Kav = (Ve – V0)/Vi
The significance of the partition coefficient can be seen from the following table:
|Partition coefficient Kav||Solute molecules are|
|0||totally excluded from the gel pores.|
|< 1||more likely to be found outside the gel pores.|
|1||distributed equally between the space inside and outside the gel pores.|
|> 1||more likely to be found inside the gel pores.|
Molecular mass estimates
For proteins that have the same general shape, globular proteins, for example, there exists a linear relationship between the molar mass Mr and the partition coefficient Kav:
Kav = -A log Mr + B
where A and B are constants.
Gel filtration chromatography resolution
The degree of separation between two peaks is called the resolution of the system used. When the peaks are poorly separated, (above, left) most of the fractions will contain a mixture of the solutes in varying proportions. A system that provides a good resolution (above, right) will effect a clean separation between the solutes.
In gel filtration, an improved resolution can be obtained by:
- decreasing the volume of sample applied to the column;
- decreasing the ratio between the sample volume and the column volume;
- increasing the column length (two or more short columns may, if practicable, be connected in series);
- correctly choosing the medium used as stationary phase (type, porosity, particle size);
- flow rate.
The range in sizes of molecules which can be separated using a given medium as stationary phase is called the selectivity of the medium. Commercially available media offer selectivities capable of separating biomolecules in ranges covering molecular masses between 102 and 8×107 daltons.
The fractionation range of a system defines the range of molecules that have partial access to the matrix pores, and thus can be expected to bes separated in that system. In order for this to occur, the Kav of the molecules should normally lie in the range 0.1- 0.7
The fractionation range of a specific gel for various types of biomolecules can normally be found in the literature supplied by the manufacturers.