ION EXCHANGE CHROMATOGRAPHY

Supervised by:Dr. Renu SolankiAssistant Professor,Department of Q.A

Submitted by:Sunil SainiM.pharm(P`ceutics) Sem-1st

LACHOO MEMORIAL COLLEGE OF SCIENCE AND TECHNOLOGY (PHARMACY WING)

JODHPUR

JAI NARAYAN VYAS UNIVERSITY, JODHPUR

In ion exchange chromatography , retention is based on the attraction between the solute ions and charged sites bound to stationary phase

Columns used for ion exchange are characterized by the presence of charged groups covalently attached to the stationary phase

Anion exchangers contain bound positive groups, where as cation exchangers contain bound negative groups

Introduction

Cationic exchangers are useful for separation of cations such as protonated bases and anion exchange columns are used for anions or acidic samples

If the stationary phase is represented by R− or R+ and the sample by X+ and X−, retention in IEC can be represented as

X+ + R−K+ X+R− + K+ (cation exchange) X- + R+Cl- X-R+ + Cl- (anion exchange)

In general , ion exchangers favour the binding of ions of

Higher charge

Decreased hydrated radius

Increased polarizability

Selectivity for ion exchange

Instrumentation

There are three classes of ion exchangers , these include

1. Resins

2. Gels

3. Inorganic exchangers

Ion exchangers

Ion exchange resins are used for the separation of small molecules.

Ion exchange gels are used for the separation of large molecules like protiens ,nucleic acids.

Separations involving harsh chemical conditions(high temperature , high radiation levels, strongly basic solutions or powerful oxidizing agents) employ inorganic ion exchangers.

Resins are amorphous particles of organic materials

Polystyrene resins for ion exchange are made by co-polymerization of styrene and divinyl benzene.

Divinyl benzene content is varied from 1 to 16 percent to increase the extent of cross linking.

Benzene groups are modified to produce cation exchange resin and anion exchange resin.

Resins

Strongly acidic cation exchanger ---sulphonic acid groups attached to styrene and di vinyl benzene copolymer.

Weakly acidic cation exchanger---carboxylic acid groups attached to acrylic and divinyl benzene co-polymer

Strongly basic anion exchanger-----quaternary ammonium groups attached to styrene and divinyl benzene co-polymer

Weakly basic anion exchanger-----poly alkyl amine groups attached to styrene and divinyl benzene co-polymer.

Classification of ion exchange resins

Sulphonate groups of strongly acidic resins remain ionized even in strongly acidic solutions , where as carboxyl groups are protonated near pH 4 and loose their cation exchange capacity

Strongly basic quaternary ammonium groups remain cationic at all values of pH, where as weakly basic tertiary ammonium anion exchangers are deprotonated in moderately basic solutions and loose their ability to bind anions

Cellulose and dextran ion exchangers , which are polymers of the sugar glucose , posses larger pore sizes and lower charge densities.

Because they are much softer than polystyrene resins , dextran and its relatives are called gels .

Ion exchange gels

Varying pH is usually a preferred way to change selectivity in ion exchange separations

An increase in the pH leads to greater sample ionization and retention in anion exchange HPLC

Eg: antibiotics containing COOH groups

Decrease in pH favours retention of bases by cation exchange HPLC

Eg: local anesthetics containing NH2 groups.

Only the ionized form of acid or base will be retained significantly

Effect of pH on ion exchange

Addition of an organic solvent to mobile phase results in decreased retention, just as in the case of reversed phase HPLC.

Solvents such as methanol or aceto nitrile are also often used in ion exchange to create changes in selectivity.

Effect of organic solvents

In ion exchange, sometimes a particular salt is selected to provide stronger or weaker retention.

A strong displacer reduces sample retention more than the same concentration of weak displacer.

In general , more highly charged displacers are stronger

Eg: relative strengths of different displacers in anion exchange chromatography F- < oxalate 2- < citrate 3-

Effect of buffers

Detectability: useful for the detection of many in-organic salts and also for the detection of organic ions with poor uv absorptivity like alkyl amines or sulfonates.

Preparative separations: usually preferred because of the availability of volatile buffers . volatile buffers makes the removal of mobile phase easier.

Useful to resolve very complex samples, i.e in the case of multi step separation

Useful for separation of mixtures of biological origin, in organic salts and some organo- metallics

Advantages

Column efficiency is less

It is difficult to achieve control over selectivity and resolution

Stability and reproducibility of the columns become questionable after repeated use.

Dis-advantages

Ion exchange chromatography is used to convert one salt to other.

Eg; we can prepare tetra propyl ammonium hydroxide from a tetra propyl salt of some other anion.

It is useful for pre concentration of trace components of a solution to obtain enough for analysis

Ion exchange is used to prepare de-ionized water

Water polishing equipment used in many laboratories uses several ion exchange cartridges.

Applications

Separation of similar ions◦ A mixture of sodium, hydrogen and potassium can be

separated using cation exchanger resin.◦ A mixture of Chloride, bromide, and iodide can be

separated using basic anion exchange resin.

Softening of hard water: Hardness of water due to cal, mg and other divalent ions. This water is passed through cation exchanger charged with the sodium ions. Ca & Mg ions retained in the column while sodium is exchanged.

APPLICATIONS OF IE CHROMATOGRAPHY

Removal of interfering radicals: Phosphate ion is the interfering with the calcium & barium ions. Phosphate is removed using sulphonic acid cation exchanger. Calcium & barium ions exchanged with H+ ions while phosphate ion pass through the column.

Separation of Lanthanides- La, Y, Ce, Rb etc

Separation of sugars: sugars-borate complexes. This complex is separated on Dewax. In this disaccharides separated from mono.

Complete demineralization of water: Removal of both cations & anions. Step A) Hard water is first passed through an acidic cation exchanger- Ca, Mg & Na are exchanged by H+ ions. Step B) This water is then passed thro a basic anion exchanger – Cl, NO2, SO4

- are exchanged by OH- ions of the exchanger.

Other applications◦For the measurement of various active ingredients in

medicinal formulations, ◦For the measurement of drugs and their metabolites

in serum and urine, for residue analysis in food raw materials,

◦For the measurement of additives such as vitamins and preservatives in foods and beverages.

Practical HPLC method development,2nd Edition, Lloyd r. snyder,pno.341-346

Instrumental methods of analysis by Willard , dean, meritt , settle, 7th edition , pno. 633-641

Principles of instrumental analysis , skoog , latest edition, pno. 641-647

References