ION EXCHANGE CHROMATOGRAPHY

1. ION EXCHANGE
CHROMATOGRAPHY

2. Hello!
I am Shruthi
I am here because I love to give
presentations.
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3. 1.INTRODUCTION

4. “ Also called ion chromatography.
 Process that allows separation of ions and
polar molecules based on their ion
exchanger.
 Charged molecules including large proteins,
small nucleotides and amino acids.
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 ION EXCHANGE RESIN--
Column filled with a charged
stationary phase on a solid
support.
 CATION EXCHAGE
CHROMATOGRAPHY--
separates out cations using
negatively charged resins.
 ANION EXCHANGE
CHROMATOGRAPHY–-
separates out anions using
positively charged resin.

6. Exchange of ions.
Two types of exchangers-
1.Cationic exchangers.
2.Anionic exchangers.
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Cationic exchangers
 Possess negatively charged group.
 Attract positively charged cations.
 Also called ACIDIC ION
EXCHANGE MATERIALS.

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Anionic exchangers
 Positively charged.
 Attract negatively charged
anions.
 Also called basic ion
exchange materials.

10. Types of ion
exchange resins
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 Two groups are used to prepare ion
exchange resins:1.Polystyrene.
2.Cellulose.
 Polystyrene resins- made by the
polymerization reaction of
styrene and divinyl benzene.
 High concentration of divinyl
benzene-produce higher cross
linkages.

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 Polystyrene resins-highly useful for separating
small molecular weight compounds.
 Cross linkage
Rigidity
Swelling
Porosity
Solubility of polymeric structure
 Sulfonic acids--strong acids
good proton dissociation ability.

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 Resins substituted with sulfonic acid groups-
strong catalytic exchangers.
 Eg for cationic exchanger-carboxymethyl
cellulose(CM cellulose).
 Eg for anionic exchanger-DEAE cellulose.

14. Preparation of
exchange medium

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1.Swelling of medium
 Makes functional groups to be
exposed for ion exchange.
 Swelling of anionic exchangers -
carried out by treating it first with
an acid(0.5 N HCl) and then with
base.

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 Swelling of cationic exchangers-matrix
is treated with EDTA for impurity
eliminations.

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2.Removal of very small
particles
༝ Fine particles-decrease the flow rate
and reaction rate.
༝ To remove these fines exchanger is
repeatedly suspended in a large
volume of water.

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 Large molecules settle down.
 Slow sedimenting materials are
decanted.

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3.Equilibrium with counter ions
 Accomplished by washing the exchangers
with different reagents depending upon the
desired counter ion to be introduced.

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NaOH
Counter ions to be introduced-”Na+”
 HCl
Counter ion to be introduced-”H+”
 NaNO3
Counter ion to be introduced-”NO3”

21. “CHOICE OF BUFFERS
 Anionic exchange chromatography-Cationic
buffers.
 Cationic exchange chromatography-Anionic
buffers.
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Buffers pH range
Ammonium acetate 4 to 6
Ammonium formate 3 to 5
Pyridinium formate 3 to 6
Pyridinium acetate 4 to 6
Ammonium carbonate 8 to 10

23. Practical
procedure
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Ion exchange separations- carried out
in columns packed with an ion
exchanger .
Ion exchangers-commercially available
made up of styrene and divinyl
benzene.
 DEAE Cellulose –anionic exchanger
CM Cellulose-cationic exchanger
 Choice of exchanger-depends upon
particle to be separated.

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 To separate anions-anionic exchangers is
used.
 To separate cations-cationic exchangers
is used.
 At first column is filled with ion
exchanger-sample is applied followed by
buffer.
 Common buffers used: Tris-buffer,
Pyridine-buffer, Acetate –buffer,
Citrate and phosphate buffers.

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 Particles which have high affinity for
ion exchanger—come down along with
the buffers.
 In the next step Tightly bound
particles are separated using
corresponding buffer.
 Then these particles are analyzed
spectroscopically.

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Advantages
 Cost effective.
 Re-usable.
 Easily collectable.
 Low maintenance cost.
 Efficient technique.
 Quick separation.

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Disadvantages
 Costly equipment.
 Expensive chemicals.
 Turbidity should be below 10
parts per million.

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Applications of ion
exchange chromatography
 Used in analysis of amino acids.
 To determine the base composition
of nucleic acid.
 Most effective method for water
purification.(softening of drinking
water)

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 Separation of proteins.
 Useful for Separation of many
vitamins, other biological amines
and organic acids & bases.

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Disadvantage
 Buffer requirements.

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WHAT TO REMEMBER!!!
༝ IEC definition.
༝ Principle.
༝ Cationic anionic exchangers.
༝ Preparation of exchange medium.
༝ Advantages.
༝ Disadvantages.
༝ Applications.

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QUESTIONS!!!!!
༝ What is ion-exchange chromatography?
༝ Write the principle of IEC.
༝ What are anionic and cationic exchangers? write an
example for each.
༝ What is pre-cycling?
༝ Write a note on preparation of exchange medium.
༝ Buffer for anionic exchange chromatography ----
༝ Buffer for cationic exchange chromatography ----
༝ What are the advantages and disadvantages of IEC?
༝ Short note on applications of IEC.

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