Chromatography is used to separate and purify bio molecules according to their difference in specific properties. This method includes a stationary phase to be purified across a mobile phase. This technique is used for the recovery, analysis, purification and separation of the products. It helps in the enrichment of target products, reduction of bulk material, removal of specific impurities, enhancement of the stability of product and prevention of product degradation. the process may be preparative or analytical. The purpose of preparative chromatography is to separate the components of a mixture for more advanced use. Analytical chromatography is done normally with smaller amounts of material and is for measuring the relative proportions of analytes in a mixture. The separation of molecules is based on difference in their structure and/or composition of molecules. In this way, different types of molecules can be separated from each other as they move over the support material.
There are several types of chromatography and they are-
i) Paper chromatography- with filter paper as a stationary phase and liquid (water, ethanol or other organic solvent) as the mobile phase. Place a small spot of the mixture to be analysed on the paper. Dip the paper in the solvent. Allow the solvent to rise up the paper. Each component dissolve int he sovent and those which are more soluble travel faster on the paper. Under similar conditions, a component should always move at the same speed. Its identity can be found by comparing the distance it moves relative to the solvent. The formula for Rf here is-
Rf= (distance travelled by the component)/(distance travelled by the solvent)
ii) Thin layer chromatography with silica as a stationary phase and a suitable organic solvent as a mobile phase. The separation technique is similar to paper chromatography. The solvent moves on the glass plate covered with silica gel.
iii) Column chromatography- The column is filled with silica as a stationary phase and liquid (organic solvent) as a mobile phase. The components interact with silica to different extent. the tap of the column is kept open to let the solvent flow out. The components travel at different rates and separate itself in different batches. The solvent in each batch is evaporated to obtain the components.
iv) High pressure liquid chromatography with silica as a stationary phase and liquid as mobile phase. This is more refined form of column chromatography. The solvent is forced to flow under high pressure. It allow faster and better purification. Both the sample and solvent are pushed through under high pressure. Different components have different retention time and the output can be detected by the component by absorbing UV light.
v) Gas liquid chromatography with silica as a stationary phase and gas as a mobile phase. A very small amount of sample is injected into the system using a syringe. The injector is contained in an oven. The boiled sample is carried into a long thin column by an inert carrier gas. The column contains a liquid stationary phase, adsorbed onto an inert solid. The time taken for the component to travel through the column to the detector. For the component, the retention time depends on the high boiling point and greater solubility.
vi) Affinity chromatography separates the biomolecules on the basis of specificity of ligands. It separates proteins on the basis of a reversible interaction between a protein (or group of proteins) and a specific ligand coupled to a chromatographic matrix. The technique offers high selectivity, hence high resolution, and usually high capacity for the protein(s) of interest. Purification can be in the order of several thousand-fold and recoveries of active material are generally very high. It has a unique characteristic in purification technology since it is the only technique that enables the purification of a biomolecule on the basis of its biological function or individual chemical structure. Purification that would otherwise be time-consuming, difficult or even impossible using other techniques can often be easily achieved with affinity chromatography. The technique can be used to separate active biomolecules from denatured or functionally different forms, to isolate pure substances present at low concentration in large volumes of crude sample and also to remove specific contaminants.
vii) Ion exchange chromatography is used for the purification of proteins, nucleotides and amino acids. This process allows the separation of ions and polar molecules based on their charge. There are two types of Ion exchange chromatography- cation exchange chromatography where positively charged molecules are attracted to a negatively charged solid support and anion exchange chromatography where negatively charged molecules are attracted to a positively charged solid support.
viii) Size Gel filtration or size exclusion chromatography involves the separation technique based on the molecular size of the components. Separation is achieved by the differential exclusion from the pores of the packing material, of the sample molecules as they pass through a bed of porous particles. The principle feature of this type of chromatography is its gentle non-adsorptive interaction with the sample, enabling high retention of biomolecular activity. It is mainly used for the separation of proteins and other water soluble polymers.
ix) Hydrophobic interaction chromatography is based on the reversible interaction between a protein and the hydrophobic ligand bound to the chromatography matrix. Hydrophobic amino acids of proteins and peptides are usually located away from molecular surfaces. However, many biomolecules have some hydrophobic groups that are sufficiently exposed to allow interaction with hydrophobic ligands on media. This is particularly suitable for samples precipitated with ammonium sulfate or eluted in high salt concentrations since high ionic strength buffers enhance the hydrophobic interaction.
x) Reversed phase chromatography purifies proteins and peptides on the basis of hydrophobicity. This type of chromatography has become quite significant for high resolution of separation and analysis of proteins, peptides, and nucleic acids.The method requires the use of organic solvents and is widely used for purity check analyses when activity and tertiary structure are not a focus. Because many proteins are denatured by organic solvents, the method is not generally recommended for preparative protein purification.
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