Gel filtration Chromatography

Gel filtration chromatography slideshare Gel Filtration is a chromatography technique used for the separation of macromolecules based on their size. Gel filtration chromatography slideshare. This technique is used to separate macromolecules with molecular weight up to 10 million (Daltons). Though it is not as efficient as high-pressure liquid chromatography and affinity chromatography, gel filtration has been found to be useful in fractionating large protein complexes. Gel filtration can also be used together with another technique called size exclusion chromatography.

Gel Filtration Chromatography:

Gel filtration is often used to separate molecules that differ in size by more than about 5% of their radius-to-molecular mass ratio. The principle behind this process is the fact that the movement of a solute through a column packed with porous beads is dependent on its radius, which in turn dictates its mobility through the pores of the beads. Thus, as a molecule passes through the smaller pores and exits at the surface, it loses confinement and can move at a significantly faster rate than if it were retained within a pore. The relative speeds of the particle and the adsorbate can be controlled by controlling the charge density on the beads.

Gel filtration Chromatography is also known as fractionation chromatography and can be further differentiated into fast and ultra-fast techniques depending on whether sample volume is less than or equal to 0.05 ml. As it utilises porous beads, gel filtration chromatography separates particles of different sizes, thereby facilitating interaction of chemical substances binding with adsorbent material. The efficiency of a technique depends greatly on the properties of active substance as slight changes in its characteristics can result in huge alterations to its mobility through porous medium.

Applications of Gel filtration Chromatography:

The uses of gel filtration chromatography are many and include the following:

1. Protein purification: Gel filtration chromatography is one of the most popular methods for protein purification. This is mainly due to its tremendous suitability in fractionating different proteins according to their molecular weight. For instance, it was used to separate two different species of immunoglobulin G (IgG) molecules having a total molecular weight of 120 kDa that were contaminated with 50 kDa heterophile antibodies potentially affecting the assays on these molecules.

Gel filtration chromatography

2. Drug synthesis: Gel filtration chromatography has been extensively applied in the field of drug synthesis. For instance, it helped separate certain antitumour drugs from different reaction mixtures prior to the characterization of their performance.

3. Gene quantification: Gel filtration chromatography can also be used to quantify certain genes by analysing their migration pattern through porous material. This requires amplification of the target gene and separation of double-stranded DNA fragments by placing them on a DNA scale in front of an appropriately designed column containing silica beads or other porous medium. The hybridised DNA fragments will move through silica beads at different speeds, thereby enabling direct quantification.

4. Enzymatic activity: Gel filtration chromatography is also used in the field of enzymatic activity, especially in the beta-lactamases industry. The column is packed with alginate beads that bind with enzymes present in a sample. The separation is based on differences of gel mobility and adsorption of different enzyme components which limit the access to the pores through which glucose enters the medium, thereby requiring higher initial pressure for cells to gain access to the pores and become active, thus increasing their initial efficiency.

5. Chromatography of organic compounds: Gel filtration chromatography serves as an effective separation technique for organic compounds ranging from simple alcohols to complex terpenes.

Gel filtration Chromatography Principle:

The principle behind gel filtration chromatography is the fact that a particular substance moves through a porous medium at different speeds depending on its molecular weight. In order to understand this concept, let us take an example of a porous medium with pores of 0.3 mm diameter and where particles must pass through it in order to reach the outlet. The inner side of the pores will absorb a substance which will move through the pores at a certain speed varying according to its size. The medium must be porous enough to allow the passage of substances of different sizes (0.2 mm, 0.4 mm and 1.0 mm) but dense enough to retain the particle thus allowing it to exit at the outlet surface.

Now let us consider a substance with a radius of 2.5 mm that is initially present in a volume of 0.25 ml in the pores. As such substance moves through the medium, the smaller size particles will absorb it by entering the pores while larger particles will remain intact within the pores until they move through at their own speed thereby rejoining with their respective size-controlling walls and continue to be bound with them. This process is repeated so the substance with a radius of 2.5 mm will only be absorbed by pores with a size of 0.3 mm.

The gel filtration chromatography principle is dependent on the fact that smaller particles will easily enter larger pores of the medium hence passing through faster than their corresponding components with larger radii. In this instance, molecules of different sizes will exit at the outlet surface in mixtures but each mix will have a set ratio between sizes. The speed at which molecules move through a porous medium is dependent on concentration and viscosity, which can be controlled by adjusting pH or temperature during sample loading and analysis.

Conclusion:

Gel filtration chromatography is a very effective technique that can easily separate different sized particles. It is extremely useful for the separation of various molecular species, especially those with similar molecular weights. It is highly recommended for use in laboratories and other fields where substances need to be separated according to their size. Gel filtration chromatography can be used for the identification of chemical compounds, drugs, enzymes and even chemical compounds commonly found in foods and beverages.