ION EXCHANGE CHROMATOGRAPHY BY G.K.Saranvijay.pptx

IEC(Ion Exchange Chromatography) is an interesting topic in instrumental methods of analysis where from intro to applications based on IEC from Chatwal book is prepared....

1. ION EXCHANGE CHROMATOGRAPHY

2. ION EXCHANGE CHROMATOGRAPHY PRINCIPLE 4 DEFINITION 3 HISTORY 2 INTRODUCTION 1 CLASSIFICATION 5 CATION EXCHANGER AND ANION EXCHANGER 6 CONTENTS

3. ION EXCHANGE METHODOLOGY 10 ION EXCHANGE CAPACITY AND EQUILIBRIA 9 MECHANISM 8 PHYSICAL PROPERTIES OF ION EXCHANGE RESINS 7 APPLICATIONS OF ION EXCHANGERS 11 CONTENTS

4. INTRODUCTION First noticed that the clay consists of metal ions in their composition and these ions has the ability to change their places with the other metal ions in their vicinity. Similar property seen in the zeolite too(Sodium Aluminium silicates). Using this phenomenon zeolite (artificial and natural) removed Ca 2+ and Mg 2+ from water. Thus providing the soft water. Ion exchangers are formed by combining a polymer and a functional groups. They are of two types namely anionic and cationic ion exchangers where the resins act as a inert support but, both types uses different functional groups.

5. HISTORY MODERN ION EXCHANGE RESINS 1935 BY ADAM AND HOLMES ABOUT ION EXCHANGE IN CLAY 1850 BY TWO BRITISH AGRICULTURAL CHEMIST ION EXCHANGE PROPERTY IN ZEOLITE 1913 BY GANS ION EXCHANGE PROPERTY IN CLAY

6. DEFINITION Ion exchange chromatography is a reversible reaction and process by which a mixture of similar charged ions can be separated by using an ion exchange resin which exchange ions according to their relative affinities. So stationary phase - synthetic- (styrene-divinylbenzene acrylic acid copolymer) ;organic - (resins, cellulose fibre, cross linked dextran [Sephadex]). Mobile phase - inorganic salt dissolved in a suitable solvent is poured or applied into the column. Examples are tris(hydroxymethyl)aminomethane, acetic acid, triethanolamine .

7. DEFINITION COMMON PROPERTIES OF STATIONARY PHASE(RESINS) AS FOLLOW : Almost insoluble in water or organic solvents like benzene, carbon tetrachloride, ether and etc., They are complex in nature. They have active or counter ion will exchange reversibly with other ions in a surrounding solutions immediately.

8. PRINCIPLE The basic principle behind this form of chromatography is attraction between the oppositely charged ions or particles. Biological substances like amino acids and proteins have ionisable group and the fact that they have net + or - ve charges can be utilised in separating of the materials. The net charge of the materials depends on the factor like pka and the pH of the solutions (Henderson- Hasselbalch equations.) Ion exchange separation are carried out in the column packed with stationary phases.

9. PRINCIPLE : 5) There are two types of ion exchangers namely Anion exchangers.(Basic ion-exchange materials). Cation exchangers.(Acidic ion-exchange materials). CATION EXCHANGERS: These poses negatively charged ions thus attracts the positive ion charges. ANION EXCHANGERS: These posers positively charged ions thus attracts the negatively charged ions .

10. CLASSIFICATION ACCORDING TO THE CHEMICAL NATURE Strong cation exchange resin - SO 3 H Weak cation exchange resin - COOH, OH ,SH , PO 3 H 2 Strong anion exchange resin - NR 3 , NR 2 . Weak anion exchange resin - NHR, NH 2 ACCORDING TO THE SOURCE They are of two types namely Natural and synthetic NATURAL: CATION - Zeolite and clay etc. ANION - Dolomite. SYNTHETIC: inorganic and organic resins.

11. FEW EXAMPLES OF CATIONIC EXCHANGERS TRADE NAME FUNCTIONAL GROUP FUNCTIONAL MATERIAL Amberlite IR - 120 -SO 3 H Styrene or divinylbenzene Dowex - SO 3 H Styrene or divinylbenzene Zerolite - SO 3 H Styrene or divinylbenzene Amberlite- 200 - SO 3 H S ame as above but heteroporous SE cellulose -C 2 H 4 -SO 3 H cellulose CM -CH 2 COOH Cellulose, fibrous Amberlite IRC -50 -COOH Methacrylic acid/divinyl

12. FEW EXAMPLES OF ANIONIC EXCHANGERS TRADE NAME FUNCTIONAL GROUP FUNCTIONAL MATERIAL Amberlite IRA - 400 -CH 2 N + (CH 3 ) 3 Styrene or divinylbenzene (homoporous) Amberlite IRA - 410 -CH 2 (C 2 H 6 )-N + -C 2 H 4 OH - Styrene or divinylbenzene (heteroporous) Zerolite FF - IP -CH 2 N + (CH 3 ) 3 Styrene or divinylbenzene (homoporous) Zerolit N-IP -CH 2 (C 2 H 6 )-N + -C 2 H 4 OH - Styrene or divinylbenzene but isoporous QAE Sephadex A - 25 -N- Dextran

13. CLASSIFICATION ACCORDING TO THE STRUCTURAL TYPE: i ) Pellicular type with ion exchange film: Size: 30-40 micro with 1-2 micro film thickness Shows very low exchange capacity and efficiency is 0.001-0.1 meq/g of ion exchange resin. ii) Porous resin coated with exchange beads Size: 5-10 micro meter and totally porous and highly efficient. Shows exchange capacity from 0.5-2 meq/g

14. CLASSIFICATION ACCORDING TO THE STRUCTURAL TYPE: iii) Macroreticular resin beads This network seen superficially on the resin bead. Not highly efficient and very low ion exchange capacities. iv)Surface sulfonated and bonded electrostatically with anion exchanger: The particles are sulphonated and bonded electrostatically with anion exchange resins. Exchange capacity is 0.02 meq/g of exchange resins.

15. ION EXCHANGERS WITH pH RANGE, NATURE AND APPLICATION CLASS OF RESIN NATURE pH RANGE APPLICATIONS ANION WEAK Formaldehyde, Polystyrene, Polyamine. 0-9 - Fractionisations of anionic complexes - Anions of different valency - Vitamins and amino acids ANION STRONG Quaternary ammonium, Polystyrene. 0-12 - Fractionisations of anions. Alkaloids, vitamins and fatty acids. CATION WEAK Carboxylic methacrylate. 5-14 - Fractionation of cations, biochemical separations, organic bases, antibiotics. CATION STRONG Sulfonated polystyrene. 1-14 - Fractionation of cations, inorganic separations and peptides and amino acids and B vitamins

16. CATIONIC EXCHANGERS It is a high molecular weight and cross linked polymer having the sulphonic, carboxylic and phenolic etc., groups as an integral part of the resin and equivalent amount of the cation and anions. In these cation exchanger and H + ions are the mobile phase where the anion remains attached to the stationary phase resin network. For example cation exchanger is kept in a solution of salt, some of the H + ions enters the solution and equivalent amount of the Na also get attached to the resin. The reaction may be represented as follow, H n R + nNa + = Na n R + nH +

17. ANIONIC EXCHANGERS An anion exchanger is a polymer having amine or quaternary ammonium groups as integral parts of the resins and an equivalent amount of anion such as Cl - ,SO 4 2- , OH - ions, etc. these ions are mobile and exchangeable. For example:

18. PHYSICAL PROPERTIES OF ION EXCHANGE RESINS PARTICLE SIZE AND POROSITY: Available in the form of fine, uniform, particle and free flow powder. Surface area directly related to the rate of exchange smaller the size of the powder greater is the surface area thus rate of exchange increases. Ion exchange are stable towards the strong acids, bases and all organic solvents. Most commonly used particles sizes range from 50-100 mesh or 100-200 mesh.

19. PHYSICAL PROPERTIES OF ION EXCHANGE RESINS CROSS LINKING AND SWELLING: More cross linking agent are present then they are rigid and swells less. When less swells the particles get trapped and cannot escape easily due to less porosity and vice versa. When swelling is more does not suit for ion exchange due to wide pores thus, optimum quantity of cross linking agents should be added to the polymeric ion exchange resin for the separation to be effective.

20. PHYSICAL PROPERTIES OF ION EXCHANGE RESINS REGENERATION: Used ion exchange resins get deactivated thus it can be used further by techniques as follow. Cation exchange resins are regenerated by using treatment of the acid solution followed by washing it in water. The resins converted into the H + form and can be used for analytical purposes. Anion exchange resins are regenerated by using treatment of the alkaline solution followed by washing with water until reaches neutral which can be used for separation of ions.

21. MECHANISM INVOLVED IN THE ION-EXCHANGERS: There are five steps and they are as follow, Diffusion of the ion to the exchanger surface. This happens so quick in homogeneous solution. Diffusion of the ions through the matrix structure of the exchangers to exchange site. This is dependent on the conc. of the solution and cross linkage of the ion exchangers. This ion exchange takes place in equilibrium manner and instantaneously. IV. Diffusion usually takes place on the surface of the ion exchanger or stationary phase.

22. MECHANISM INVOLVED IN THE ION-EXCHANGERS: V. Selective desorption of the eluant through the diffusion to the external solution which is based on the factors like pH, ionic concentration and by affinity elution. NOTE: More highly charged ions to be exchanged greater is tightly bound to the surface of the stationary phase. The rate of the ion exchange is controlled by the law of mass action. Usually the greater ionic metal always remove the functional group. For example the above illustrated diagram shows that sodium replaces the hydrogen (acid) from the stationary phase but if incase all the acid content is washed away then the solution becomes more concentration and the process reverses ie., the Na + replaced by the H + .

23. MECHANISM: CATION EXCHANGER: + + STATIONARY PHASE WITH Na + + CHARGED MOLECULE TO BE EXCHANGED = CHARGED ION BOUND WITH STATIONARY + EXCHANGED PHASE ION ANION EXCHANGER: + + STATIONARY PHASE WITH Cl - + CHARGED MOLECUE TO BE EXCHANGED = CHARGED ION BOUND WITH STATIONARY + EXCHANGED PHASE ION RSO 3 - N + H 3 R ’ Na + RSO 3 - Na + N + H 3 R ’ (R) 4 N + COO - R + Cl - R 4 N + Cl - COO - R +

24. MECHANISM In general at equal concentration of metals and functional group always replaced by the metals i.e., the site of the functional group is taken by the metal group but the anion remain attached to the resins. The order of affinity towards the any functional group are as follows: The ions with the highest charge has the higher affinity that is Na + <Ca 2+ <Al 3+ . Down the column of the atomic table ie size and charge of the atom increases thus affinity also increases Li + <Na + <K + <Cs + <Be 2+ <Mg 2+ <Cu 2+ <Sn 2+ If the ion becomes too large then the distortion of the resin takes place and rules break down.

25. ION EXCHANGE EQUILIBRIA Ion exchange resins contains fixed charge o matrix and counter balance opposite replaceable charge. The replaceable charge in the solution is readily exchanged with the charge on resin. If the solution contains high number of C + , D + , E + etc. The exchanger shows different affinity for the cations. It is observed that exchange the higher valent molecule for lower valent molecule is favoured by increasing the concentration. If incase exchange of lower valent molecule for higher valent molecule is favoured by dilution.

26. ION EXCHANGE EQUILIBRIA Governing factors for exchange of ions are as follow: Nature of ion exchange resins i.e., strong or weak type. Nature and number of functional groups on resin. pH of the solution. Concentration of solution in contact with resin.

27. ION EXCHANGE CAPACITY It is total ion exchange capacity in terms of the exchangeable functional groups are expressed in milliequivalents per gram of the ion exchange resins. meq/g = 1000/equivalent weight

28. METHODOLOGY: COLUMN: Columns used in the laboratories are made up of glass whereas in industries high quality stainless steel or polymer column were used which can withstand strong acids and alkali. It should be mechanically stable. The geometry of the column depends on separation factor. Separation can be improved by increasing the length of the column but only to a certain. Generally the ratio of column can be 10:1 or 100:1 of height to diameter.

29. METHODOLOGY: TYPE OF ION EXCHANGE RESIN AND PHYSICAL CHARACTERISTICS: Selection of the ion exchange resin depends on the following factors: Type of the ion to be separated - cation or anion. Nature of the ions to be separated - strong or weak. Efficiency of the resin - capacity. Particle size of the resin - 50 - 100 mesh or 100 - 200 mesh. Structural type of the resin - porous or pellicular. Amount of the cross linking agents - optimises the quantity used.

30. METHODOLOGY: MOBILE PHASE: Basically the mobile phase depends on the factors like strength of the acids, alkali and buffers are used as eluting solvents.(example: 0.1 N HCl, 1N NaOH, phosphate buffer, acetate buffer and etc.) and organic solvents are not used in the ion exchange chromatography. PACKING THE COLUMN: The resin treated with solvent and attained equilibria before packaging of the column. The slurry poured into the column and solvent used for the eluent should be used in the slurry. The slurry added in several way allowing the resin to settle down. To attain constant flow rate pour the solvent into the column.

31. METHODOLOGY: PACKING THE SOLVENT: The level of solvent adjusted. APPLICATION OF THE SAMPLE: After packing the column the solution poured or injected into the column. This is allowed to the bed of ion exchanger. For this purpose the syringe or pipette is utilised. And sometime allowed to be in the contact with the ion exchangers ELUTION: The components of mixture separate and move down the column individually at different rates depending upon the affinity of the ion for ion exchanger. The ions with least attraction will move most rapidly with the solvent and as they move downwards. The distance between them increases.

32. METHODOLOGY: The eluates are collected at different stages. The efficiency of separation increases with increasing column length and low flow rates. ANALYSIS OF ELUATE Different fractions collected with volume or time is analysed for their contents. The eluate collected after passing through ion exchange column and analysed by various methods depends upon nature and quantity of the sample. Analysis methods such as spectroscopic methods, polarography, radiochemical methods refractive index, pH, light absorption etc. The graph plotted against reading and volume of eluate and results calculated.

33. METHODOLOGY:

34. FACTORS AFFECTING ION EXCHANGE SEPARATIONS : The factors affecting ion exchange separations are: Nature and properties of ion exchange resins Nature of exchanging ions ION EXCHANGE CHROMATOGRAPHY FACTORS AFFECTING ION EXCHANGE SEPARATIONS Nature of ion exchange resin Crosslinking and swelling is import factor which depends on the proportion of cross linking agent (divinylbenzene) and polystyrene. When more cross linking agent is Present, they are more rigid, but swell less. When swelling is less, separation of ions of different sizes is difficult as they cannot pass through the pores present and it becomes selective-toions of different sizes.

35. FACTORS AFFECTING ION EXCHANGE SEPARATIONS: When less cross linking agent is present, they are less rigid, but swell more. When swelling is more, separation will not be sufficient as exchange of functional groups does not take place due to wide pore. Hence an optimum quantity of cross linking agent should be added to the polymeric ion exchange resins for the separation to be effective. Nature of exchanging lons I. Valency of ions: At low concentrations and at ordinary temperatures, extent of exchange increases with increases with increase in valency Na + < Ca 2+ < Al 3+ < Th 4+ II. Size of ions: For similar charged ions, exchange increases with decrease in the size of hydrated ion. Li + <H + < Na + < NH 4+ < K + < Rb + <Cs +

36. FACTORS AFFECTING ION EXCHANGE SEPARATIONS: III. Polarizability: Exchange is preferred for greater polarizable ion I - < Br - < Cl - < F - IV. Concentration of solution: In dilute solution, polyvalent anions are generally adsorbed preferentially. V. Concentration and charge of ions: If resin has higher +ve charge and solution has lower +ve charge, exchange is favoured at higher concentration. If the resin has lower +ve charge and solution has high +ve charge, then exchange is favoured at low concentration.

37. APPLICATIONS: 1. Separation of similar charged ions Ion exchange chromatography is used for separation of similar charged ions. Mixture of H+, Na+, and K+ can be separated by using cation exchange resin. Similarly Cl, Br, I can be separated by passing through basic anion exchanger. 2. Demineralisation of water Demineralisation of water requires removal of ions i.e. cations as well as anions. Water is passed through an acidic cation exchanger when metallic cations are exchanged with H+ ions. This water obtained is then passed through a basic anion exchanger when the anion present in the water exchanged by OH- of the exchanger. The H+ and OH- ions which pass into the solution combine to form unionized water. Generally sulfonic acid resin is used as a cation exchanger while strong basic resin is employed as an anion exchanger.

38. APPLICATIONS: Water is passed through an acidic cation exchanger when metallic cations are exchanged with H+ ions. This water obtained is then passed through a basic anion exchanger when the anion present in the water exchanged by OH- of the exchanger. The H+ and OH- ions which pass into the solution combine to form unionized water. Generally sulfonic acid resin is used as a cation exchanger while strong basic resin is employed as an anion exchanger. 3. Softening of hard water Hard water passing cation exchanger charged with Na+, Cat and Mg+ ions from water are retained on the column while Na+ ions pass into the solution. 4. Separation of sugars Sugars are first converted into borate complex and the separation of borate complexes of sugar have been achieved quantitatively on column of Dowex. In this, Disaccharides can be separated from monosaccharides and individual compounds of hexose pentose from mixture can be resolved. 5. Purification and recovery of pharmaceuticals This process used for purification and recovery of antibiotics, vitamins, alkaloids, hormones and other chemicals of pharmaceutical importance during their manufacture.

39. APPLICATIONS: 6. Medicinal importance Anionic resins are introduced in the treatment of ulcer while cation exchangers have been used to remove Na+ from body during the treatment of hypertension and oedema. Resins are also used as a diagnostic aid in gastric acidity tests. The resins have been used with other medicinal agent to achieve delayed action dosages. 7 . Organic separations Most of the pharmaceutical coin either strongly or weakly acidic or basic in nature. Hence of those compounds can be separated by using ion exchange So which can be separated are amino proteins, antibiotics, vitamins, fatty acids, etc. example, a mixture of acidic, neutral and basic amino acid can be separated using ion exchange column. Similarly a inbal: of vitamins like Vitamin B1, B2, nicotinic acid, folic acid; cyanocobalamin etc., can be separated using ion exchange technique. 8. Biochemical separations Like isolation of some drugs or metabolites from blood, urine etc.

40. APPLICATIONS: 9. Ion exchange column in HPLC For separation of compounds of mixed nature like acidic and basic substances, ion exchange column is used in HPLC (High performance liquid chromatography). 10. Ion exchange in purifying the radioactive wastewater from industries: