Ion exchange chromatography

by ajesh unnikrishnan college of pharmaceutical sciences trivandrum

1. WELCOME

2. ION EXCHANGE CHROMATOGRAPHY PRESENTED BY AJESH U FIRST SEM M-PHARM PHARMACUETICAL CHEMISTRY CPS GOVT MEDICAL COLLEGE THIRUVANANTHA PURAM

3. DEFINITION Reversible reaction in which free mobile ions of an ion exchanger are exchanged with similar charge present in solution Used for large proteins, small nucleotides and amino acids Ion exchangers are insoluble organic polymer where charged groups are introduced styrene- divinyl -benzene copolymers or its acrylic acid derevatives are used as back bone For biological macromolecules celluose fibers is used Cross-linked dextran ( sephadex ) has been also used

4. PROPERTIES OF ION EXCHANGERS Insoluble in organic solvents or water ( eg ; benzene, carbon tetrachloride, ether etc…) Complex ( ie ; polymeric) Swollen resin will be denser than water Chemically stable Have active or counter ions

5. PRINCIPLE Biological molecules have net positive or negative charge T he net charge is due to pK a and pH Ion exchangers have active ions to attract oppositely charged ions

6. ION EXCHANGE MECHANISM Diffusion of ion to exchanger surface Diffusion of ion through matrix site of exchanger to exchange site . depended up on cross linkage of the exchanger and concentration of solution Hence controls the rate of whole ion exchange process 3) Exchange of ions at exchange site

7. Anion exchanger Cation (R) 4 N + ----- Cl - + - OOCR (R) 4 N + ----- - OOCR + Cl - RSO - 3 ------NA + + N + H 3 R RSO - 3 ------ NH 3 R + NA + Diffusion of exchanged ion Desorption by the eluant

8. ION EXCHANGERS BASED ON SOURCE NATURAL; cationic – zeolytes,clay anion – dolomite SYNTHETIC RESINS; inorganic and organic BASED ON CHEMICAL NATURE Anion exchange Cation exchange

9. CATION EXCHANGERS Polymeric anion to which active cations are attached Cations can be sulphonic , carboxilic , phenolic etc… H ydrogen ions are mobile and exchangeble Anions like –coo - , so 2- -o - attach to the resin

10. ANION EXCHANGER Polymeric cations to which active anions are attached Anions can be amine , quaternary ammonium groups etc… Cl - , SO 4 2- , OH - are mobile and exchangeble Anions like –CH 3 – N + bind to the resin

11. REGENERATION Cation exchange resin are regenerated by treatment with acid and washing with water Anion exchange resin are regenerated by treatment with NaOH , then washing with water untill neutral This step enhances its efective capacity

12. BUFFER pH of the mobile phase must be between and pKa I n cation exchange if pKa =1.2 and iso electric point=8.2 then mobile phase pH can be 6 In anion exchange if pKa =10.3 and iso electric point = 6.8 then mobile phase pH can be 8.0

13. ALTERATION OF pH Cation exchange – rasing pH – molecule become less protonated – easily eluted Anion exchange – lowering pH – molecule become more protonated – easily eluted After the alteration of pH the molecule can not form ionic interaction

14. ION EXCHANGE COLUMN Different ions have different capacity to undergo exchange In cations ; Th 4+ > Al 3+ > Ca 2+ > Na + In anions; PO 4 3- > SO 4 2- > I - In ions carrying same charge size of hydrated ion is considered H + >Cs + > Rb + etc…

15. SELECTION OF SUITABLE SYSTEM SPECIFICATION IONIC GROUPS SUITABLE MEDIUM STRONG ACID SO 3 - ACIDIC AND ALKALINE WEAK ACID COO - ONLY ALKALINE STRONG BASE N + R 3 ACIDIC AND ALKALINE WEAK BASE NHR 2 ONLY ACIDIC

16. ION EXCHANGE CAPACITY Characterised by number of ion active groups Expressed in terms of milliequivalents /g of ion exchange resin In cationic exchanger Alkali ( NaOH ) required to titrate all acid from resin C= capcity V=volume of alkali N=strength of alkali W= wegit of resin In anion exchanger sodium nitrate is passed and calculated

17. ION EXCHANGE TECHNIQUES 1)BATCH METHOD Resin and solutions are mixed in a vessel until equilibrium is attained Used for softening of water 2)COLUMN METHOD Diference in selectivity co efficient leads to diference in separation Consist of glass column with wool plug at lower end and slurry is poured in to it

18. INSTRUMENTATION

19. STEPS INVOLVED EQUILIBRATION LOADING WASHING ELUTION

20. GRAPHICAL INTERPRETATION Time in X axis Absorbance in Y axis Peak area is measured for concentration of sample

21. APPLICATIONS 1)Separation of lanthanides and actinides 2)Softening of water

22. 3) Seperation of amino acids

23. 4) Seperation of similar ions from other 5) Removal of interfering radicals 6) Demineralisation of water 7) Purification of organic compounds extracted in water 8) Prepration of pure reagents 9) hydrometallurgy

24. BE WISE REVICE Definition Principle and mechanism Ion exchangers Regeneration, buffer, alteration of pH Ion exchange column Selection of suitable system Ion exchange capacity Ion exchange technique Instrumentation Steps involved Graphical interpretation applications

25. REFERENCE Instrumental methords of chemical analysis by gurdeep R chatwal and sham K anand ; pp;2.662-2.672 Instrumental analysis by skoog,holler and crouch; pp;918-923

26. THANK YOU