2. Introduction
• 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
3.
4. • Cationic exchangers are useful for separation of cations
such as protonated bases and anion exchange columns
are used for anions or acidic samples
5. • 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)
6.
7. Selectivity for ion exchange
• In general , ion exchangers favour the binding of ions of
• Higher charge
• Decreased hydrated radius
• Increased polarizability
9. Ion exchangers
• There are three classes of ion exchangers , these include
1. Resins
2. Gels
3. Inorganic exchangers
10. • 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
11. Resins
• 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
12.
13. Classification of ion exchange 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
14. • 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
15. Ion exchange gels
• 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 .
16. Effect of pH on ion exchange
• 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
17. Effect of organic solvents
• 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.
18. Effect of buffers
• 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-
19.
20. Advantages
• 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
21. Dis-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.
22. Applications
• 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.
23. APPLICATIONS OF IE CHROMATOGRAPHY
• Separation of similar ions
o A mixture of sodium, hydrogen and potassium can be separated using cation
exchanger resin.
o 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.
24. • 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.
25. • 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.
26. • 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
o For the measurement of various active ingredients in medicinal formulations,
o For the measurement of drugs and their metabolites in serum and urine, for
residue analysis in food raw materials,
o For the measurement of additives such as vitamins and preservatives in foods
and beverages.
27.
28. References
• 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
