ION EXCHANGE CHROMATOGRAPHY
Created By…..Desai Chandni
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. 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.
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.
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.
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.
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.
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.
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