DNYANASADHANA COLLEGE, THANE ION EXCHANGE CHROMATOGRAPHY By Dr.Bhagure G.R. DNYANASADHANA COLLEGE, THANE

ION EXCHANGE CHROMATOGRAPHY ADSORPTION Stationary phase Ion exchange Resin Liquid Mobile phase ADSORPTION

ION EXCHANGE CHROMATOGRAPHY Proteins, Poly Peptides Nucleic Acids Nucleotides Charged Molecules ION EXCHANGE CHROMATOGRAPHY

Ion Exchange Ion exchange is an adsorption phenomenon where the mechanism of adsorption is electrostatic. Electrostatic forces hold ions to charged functional groups on the surface of the ion exchange resin. The adsorbed ions replace ions that are on the resin surface on a 1:1 charge basis. For example:

Na+ H+

The first ion exchangers were synthetic resins designed for Applications such as demineralization, water treatment, and recovery of ions from wastes. The first ion exchangers designed for use with biological substances were the cellulose ion exchangers developed by Peterson and Sober. Ion exchangers based on dextrin (Sephadex), followed by those based on agarose (Sepharose CL-6B) and cross-linked cellulose (DEAE Sephacel) were the first ion exchange matrices to combine a spherical form with high porosity, leading to improved flow properties and high capacities for macromolecules

THE ION EXCHANGE RESINS Polymeric resins are made in 3-D networks by cross-linking hydrocarbon chains. The resulting resin is insoluble, inert and relatively rigid. Ionic functional groups are attached to this framework.

INTEGRAL PART

Ion Exchange Resin:

CHARACTERISTICS OF THE ION EXCHANGE RESINS Ion exchange resin should have following characteristics. It should be insoluble in aqueous medium. Should be denser than water Should have lose porous polymeric structure. It should be inert in nature Should have large exchangeable sites Should have high degree of cross linking.

CLASSIFICATON OF ION EXCHANGE RESINS CATION EXCHANGE RESINS STRONGLY ACIDIC CATION EXCHANGE RESIN WEAKLY RESIN ANION BASIC ANION

STRONGLY ACIDIC CATION EXCHANGE RESIN R SO3H

WEALKLY ACIDIC CATION EXCHANGE RESIN R COOH

(Integral part) (Labile ion) STRONGLY BASIC ANION EXCHANGE RESIN R QAS OH- Ex. R-(NCH3)3+ Cl-- (Integral part) (Labile ion)

R OH- R- Primary amino group or secondary amino group WEAKLY BASIC ANION EXCHANGE RESIN R OH- R- Primary amino group or secondary amino group

Factors affecting separation of Ions: The charge on the ion. The size on the ion . The concentration of the ion Use of complexing agent and ability to form complexes Nature of the resin. Effect of pH.

1) The charge on the ion: Attraction of the resin for an ion increases with increase in the charge carried by the ion. The preference of the resin can be follow the order. Ex. Th4+ >Al3+, >Ca2+>Na+

Resin Less affinity LargeSize Small Size More affinity The size on the ion : The size of the bare ion is not considered here because ions get hydrated when they are in the aqueous medium. The ions having small size get hydrate more and have less affinity with resin and vice versa i.e. The ions having large size get hydrate less and have more affinity with the resin. Ex. Li+ <H+ <Na+< NH4+ < K+<Rb+ Less affinity LargeSize Resin Small Size More affinity

3)The concentration of the ion: Higher the concentration of the ion (same ion) in the solution higher is the affinity of the ion with the resin.

4)Use of complexing agent and ability to form complexes : The ion having ability to form complexes its size will increases and its affinity with the resin is decreases and vice versa. 5) Nature of the resin. Resin having spherical shape, high degree of cross linking, strength of functional group decides its ability for the exchange of ions. 6) Effect of pH : The pH can affect on separation by several ways, The ionization of the resin in solution depends on the pH especially if the functional group is weakly acidic or basic. The strength of acids or bases, the hydrolysis of the salts, the ionization of the functional group of the resin, all are strongly dependent on pH of the solution. For better ion exchange, weakly acidic anion exchanger has to used in alkaline medium. Similarly weakly basic anion exchange has to use in acidic medium

ION EXCHANGE CAPACITY Efficiency of ion exchange process depends upon exchange capacity of resin. The number of mill equivalent ion exchange by one gram of dry resin is called as ion exchange capacity. One gram of cation exchange resin is soaked in HCl acid to convert the resin in H+ form. It is then placed in water. The glass tube like that of burette is packed with the resin. 10 ml of 0.5N solution of Na2SO4 is placed on the resin column. The elution is carried out using distilled water. The eluate is collected in conical flask and then titrated with 0.1N NaoH solution using phenolphthalein indicator. Volume of NaoH required for the titration is found out and cation exchange capacity is determined by using following formula. VxN Cation exchange Capacity: ---------- W V: - Volume of Burette reading. N: - Normality of NaoH W: - weight of dry resin

Anion Exchange Capacity One gram of anion exchange resin is soaked in HCl acid to convert the resin in cl- form. It is then placed in water. The glass tube like that of burette is packed with the resin. 10 ml of 0.5N solution of Na2SO4 is placed on the resin column. The elution is carried out using distilled water. The eluate is collected in conical flask and then titrated with 0.1N AgNo3 solution using Potassium chromate indicator. Volume of AgN03 required for the titration is found out and anion exchange capacity is determined by using following formula. VxN Cation exchange Capacity: ---------- W V: - Volume of Burette reading. N:- Normality of AgNo3 W: - weight of dry resin

EXPERIMENTAL TECHNIQUE Water level Ion Exchange Resin Cotton Plug Or glass wool plug

Elution:-The process of removing adsorbed ions is known as elution. Terms involved Elution:-The process of removing adsorbed ions is known as elution. Eluent :-The solution used for elution is called as eluent . Eluate.:- The solution resulting from the elution is called as eluate.

APPLICATION OF ION EXCHANGE CHROMATOGRAPHY. PREPARATION OF DEMINERLISED WATER OR DEIONISED WATER. Separation of Lanthanides. Separation of amino acids. Determination of concentration of trace constituent. Separation of similar ions. Separation of interfering cations and anions.

1) PREPARATION OF DEMINERLISED WATER OR DEIONISED WATER. Water from which all cations and anions are removed is called as Deminerlised water or deionised water. The process involves two steps. a) In the first step water is passed through the cation exchange resin column (H+ form). While passing through the column all cations such as Na+, Mg2+ are exchanged for H+ ion . R-SO3‑H+ + M+ --------- RSO3- M+ ----------- b) In second step, water coming out from first column is allowed to pass through an anion exchanger in basic form (OH-) In this anions like Cl- ,SO42- are exchanged for OH-. R- OH- + Cl-+ --------- R--Cl- + OH-. H+ ions combine with OH- to give unionized water.

2) Separation of Lanthanides:- In this column is packed with cation exchange resin of type R-H. When solution containing mixture of lanthanides is poured on the top of the hen following equilibrium takes place, 3R-H + Ln3+ ---------------- R3 ____Ln 3+ 3H+ <-------------- In case of lanthanides the ionic radii of tripositive ions decreases with increase in atomic number i.e. from lanthanum to lutecium. Hydration of ion increases with decrease in ionic size and therefore hydrated size of lanthnone ion increases with increase in atomic number. Lanthnone ion having small hydrated size( La) will strongly adsorb on the resin and vise versa (Lu). Hence lanthnone ion held by resin decreases from La to Lu. When HCL solution is passed as mobile phase separation of lanthanides takes place in reverse order of atomic number. Lutecium ion will separate out first where as Lanthanum ion separate at last. For better separation elution is carried out using ammonium Citrate buffer at pH 2.8 -3.4.

3) Separation of amino acids:- Chromatographic column is packed with cation exchange resin. Solution of metal ions like Cu2+,Cd2+ are exchanged on the column. When amino acid mixture passed on the column it forms complexes with varying stability. Most stable complex will elutes first and least stable will elutes at last. Determination of concentration of trace constituent:- Trace amount substance present in large volume can be found out. Ex. If trace amount of Ag +ion is present in large volume of water .In this Column is packed with Cation exchange resin. Then solution is passed. Ag +ion exchange with cation. Silver is then eluted by using small amount of eluent.

5) Separation of similar ions. Ex. Similar ions like Cl-,Br- and I- can be separated using ion exchange chromatography. Column is packed with anion exchange resin like R-OH- .When solution containing halides passed over the column these anions will adsorb at various extent. When elution is carried out using 0.3M NaNo3 Solution Cl-will elutes out first. When concentration NaNo3 increases to 0.6M After elution Br - ion eluted similarly I- ion eluted at last using 0.9M NaNo3. 6) Separation of interfering cations and anion:- Interfering ion can be removed by ion exchange chromatography. Ex.In the estimation of Ca2+ and Mg2+ion by by sulphate method, Phosphate ion interfere in the estimation. This Phosphate ion can be removed by passing solution through cation exchanger, Ca2+ and Mg2+ions get exchanged for H+ ion. While phosphate ion s pass through exchanger. Ca2+ and Mg2+ions held by resin are eluted and then estimated.

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