2. Desai Chandni

3.  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

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

6.  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)

8.  In general, ion exchangers favour the binding of ions of  Higher charge  Decreased hydrated radius  Increased polarizability

10.  There are three classes of ion exchangers, these include 1. Resins 2. Gels 3. Inorganic exchangers

11.  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

12.  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

14.  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

15.  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

16.  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.

17.  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 NH 2 groups.  Only the ionized form of acid or base will be retained significantly

18.  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.

19.  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-

21.  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

22.  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.

23.  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.

24.  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.  METHOD: Mixture of chloride, bromide & iodide is passed through basic anion exchanger using 0.5M sodium nitrate as eluant. Chloride will first elute. Raise the conc of Sodium Nitrate, Bromide will elute, raise the conc of Sodium Nitrate further, iodide ion will elute.

25.  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.  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.

26.  Complete demineralization of water: Removal of both cations & anions. Step A) Step A) Hard water is first passed through an acidic cation exchanger- Ca, Mg & Na are exchanged by H + ions. Step B) Step B) This water is then passed thro a basic anion exchanger – Cl, NO 2, SO 4 - are exchanged by OH - ions of the exchanger.  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.

27.  Separation of Amino Acids: protein after hydrolysis is introduced to a short column on special polystyrene sulphonic acid resin at pH 2 and eluted with 0.35N sodium citrate buffer of pH 5.25. acidic & neutral AAs first leave the column as unseparated then others. pH 2  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.

29.  Practical HPLC method development,2 nd Edition, Lloyd r. snyder,pno.341-346  Instrumental methods of analysis by Willard, dean, meritt, settle, 7 th edition, pno. 633- 641  Principles of instrumental analysis, skoog, latest edition, pno. 641-647