Ion exchange chromatography uses charged sites on a stationary phase to retain ions from a solution based on electrostatic attraction. There are three main types of ion exchangers: resins, gels, and inorganic exchangers. Resins are the most common and are made from organic polymers like polystyrene cross-linked with divinylbenzene. The charged groups can be cationic like sulfonic acid or anionic like quaternary ammonium. Ion exchange chromatography is useful for separating ions and ionizable compounds based on factors like charge and size.

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

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 NH2 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 inorganic 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) 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.

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.
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,2nd
Edition, Lloyd r. snyder,pno.341-346
Instrumental methods of analysis by Willard ,
dean, meritt , settle, 7th edition , pno. 633641
Principles of instrumental analysis , skoog ,
latest edition, pno. 641-647