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Ion exchange chromatography
Ion exchange chromatography separates ions and polar molecules based on their affinity for an ion exchange resin. It works through the reversible electrostatic interaction between ions in solution and ions attached to the resin. There are four main types of resins: strong cation, weak cation, strong anion, and weak anion. Organic resins like polystyrene with divinylbenzene crosslinking are commonly used. The process involves equilibrating, applying the sample, eluting components at different rates depending on their affinity, and regenerating the resin. Ion exchange chromatography has applications like water softening, enzyme purification, and separation of ions, sugars, amino acids and proteins.
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Ion exchange chromatography
- 1. ION EXCHANGE CHROMATOGRAPHY By M.Vharshini B.Sc.Bio Medical Science Sri Ramachandra University
- 2. Ion-exchange chromatographyis a process that allows the separation of ions and polar molecules based on their affinity to the ion exchanger. It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids. Cations or Anions can be separated using this method. PRINCIPLE It is based on the reversible electrostatic interaction of ions with the separation matrix (i.e.) The separation occurs by reversible exchange of ions between the ions present in the solution and those present in the ion exchange resin.
- 3. CLASSIFICATION OF RESINS Accordingto the chemical nature they classified as- 1. Strong cation exchange resin 2. Weak cation exchange resin 3. Strong anion exchange resin 4. Weak anion exchange resin
- 4. Natural resins: Cation - Zeolytes, Clay Anion - Dolomite Synthetic resins: Inorganic & Organic resins ◘Organic resins are polymeric resin matrix. The resin composed of – Polystyrene (sites for exchangeable functional groups) Divinyl benzene(Cross linking agent)-offers stability.
- 5. »It must bechemically stable. »It should be insoluble in common solvents. » It should have a sufficient degree of cross linking. »The swollen resin must be denser than water. »It must contain sufficient no. of ion exchange groups.
- 6. Cross linking: It affectsswelling & strength & solubility Swelling: When resin swells, polymer chain spreads apart Polar solvents → swelling Non-polar solvents → contraction Swelling also affected electrolyte concentration.
- 7. Particle size andporosity Increase in surface area & decrease in particle size will increase the rate of ion exchange. Regeneration Cation exchange resin are regenerated by treatment with acid, then washing with water. Anion exchange resin are regenerated by treatment with NaOH, then washing with water until neutral.
- 12. 1.Column » glass, stainlesssteel or polymers 2.Packing the column » Wet packing method: A slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles. 3.Application of the sample After packing, sample is added to the top of the stationary phase, use syringe or pipette. This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluent.
- 13. 4.Mobile phase Acids, alkalis,buffers… 6.Stationary phase The ionic compound consisting of the cationic species (M+) and the anionic species (B-) 7.Elution Components of mixture separate & move down the column at different rates depending upon the affinity of the ion for ion exchanger. The eluates are collected at different stages 8. Analysis of the eluate Spectrophotometric, flame photometry polarographic, conductometric…
- 16. a. Nature &properties of ion exchange resins: Cross linking & swelling is important. If more cross linking , they are more rigid but swelling is less. swells less → separation of ions of different sizes is difficult. b. Nature of exchanging ions: 1. valency of ions. 2.Size of ions.
- 17. 3.Polarizability 4.Concentration of solution. 5.Concentration& charge of ions. c. pH of the mobile phase d. Ionic strength e. Mobile phase modifiers f. Temperature
- 18. The basicprocess of chromatography using ion exchange can be represented in 4 steps: 1. Equilibration 2. Sample application and wash 3. Elution 4. Regeneration
- 25. APPLICATIONS ◘ softening anddemineralisation of water. ◘ For extraction of enzymes from tissues. ◘ purification of solutions free from ionic impurities. ◘ separation of inorganic ions. ◘ separation of sugars, amino acids and proteins. ◘ ion exchange column in HPLC.
- 26. Himmelhoch, SR(1971) Chromatography of proteins on ion-exchange adsorbents. Meth. Enzymol. 22:273-286. Scopes, RK (1982) Ion exchangers-principles, properties and uses. In “Protein www.harvardapparatus.com/ion exchange chromatography http://machias.edu/basic-principles-of-ion- chromatography.html