Purification techniques chromatography

Purification techniques chromatography

1. Purification T echniques:Chromatography Mr.Halavath Ramesh Department of chemistry Loyola College –Chennai University of Madras E-Mail: [email protected]

2. Introduction Biomolecules are purified using purification techniques that separate according to differences in specific properties . Property Technique 1. Bio-recognition( Ligand specificity) …….Affinity Chromatography 2. Charge ……..Ion exchange chromatography 3. Size …..Gel filtration (some times called size exclusion) 4. Hydrophobicity …… Hydrophobic interaction chromatography, Reversed phase chromatography

3. Affinity Chromatography Affinity chromatography separates proteins on the basis of a reversible interaction between a protein ( or group of proteins) and a specific ligand coupled to a chromatography matrix. Affinity chromatography is unique in purification technology since it is the only technique that enables the purification of a biomolecules on the basis of its biological function or individual chemical structure. 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.

4. Ion exchange chromatography Ion exchange chromatography is a techniques that is commonly used in bio-molecule purification. It involves the separation of molecules on the basis of their charge. This technique exploits the interaction between charged molecules in a sample and oppositely charged moieties in the stationery phase of the chromatography matrix. This type of separation is difficult using other techniques as charge is easily manipulated by the pH of buffer used. Two types of ion exchange separation is possible - Cation exchange and anion exchange.  In anion exchange the stationary phase is positively charged whilst in Cation exchange it is negatively charged. It involves the four stages 1. Equilibrium 2. Sample application and wash 3. Elution 4. Regeneration. In ion exchange chromatography adsorption and desorption processes are determined by the the properties of the three interaction entities: 1. The stationary phase 2. Mobile phase( Eluent ) 3.The solute.

5. Size exclusion chromatography Size exclusion chromatography (SEC) also called gel-filtration or gel-permeation chromatography(GPC) uses porous particles to separate molecules of different size. When an aqueous solutions is used to transport the sample through the column the techniques is known as Gel filtration chromatography. A mixture of molecules dissolved in liquid (the mobile phase ) is applied to a chromatography column which contains a solid support in the form of microscopic spheres or beads( the stationary phase). Buffer: The liquid used to dissolve the biomolecules to make the mobile phase is usually called a Buffer. Still , the use of the hydrodynamic volume, a size based on dynamical properties, in the interpretation of SEC data is not fully understood . This is because SEC is typically run under low flow rate conditions where hydrodynamic factor should have little effect on the separation.

6. Reverse phase chromatography Reversed –phase chromatography (also called hydrophobic chromatography). Reversed-phase chromatography is a technique using alkyl chains covalently bonded to the stationary phase particles in order to create a hydrophobic stationary phase, which has a stronger affinity for hydrophobic or less polar compounds. The use of a hydrophobic stationary phase is essentially the reverse of normal phase chromatography, since the polarity of the mobile and stationary phases have been inverted – hence the term reversed-phase chromatography . Reversed-phase chromatography employs a polar ( aqueous ) mobile phase. As a result, hydrophobic molecules in the polar mobile phase tend to adsorb to the hydrophobic stationary phase, and hydro philic molecules in the mobile phase will pass through the column and are eluted first.

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