Ion exchange chromatography is a process that separates ions and polar molecules based on their charge using an ion exchange resin. There are two main types of ion exchange - cation exchange which uses a negatively charged resin to adsorb positively charged proteins, and anion exchange which uses a positively charged resin to adsorb negatively charged proteins. The process involves equilibrating the resin, applying the sample mixture, then eluting the bound molecules by altering conditions such as pH or ionic strength to cause differential elution. Ion exchange chromatography is useful for purifying proteins and other charged biomolecules.

1. Ion exchange chromatography
Submitted by: Humna Mehmood
BS Chemistry (2017-2021)
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2. Contents
Introduction
Principle
Classification of ion exchange resin
Requirements of ion exchange resin
Selection of resin
Practical requirements of IEC
Steps in ion exchange chromatography
Methods of ion exchange chromatography
Chromatographic parameters
Factors affecting ion exchange chromatography
Ion exchange constant
Applications of ion exchange chromatography
Advantages of ion exchange chromatography
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3. Ion exchange chromatography
Introduction:
Ion exchange chromatography is a process by which a mixture
of similar charged ions can be separated by using an ion-
exchange resin which exchanges ions according to their relative
affinities.
It is a process that allows the separation of ions and polar
molecules based on their charge.
A form of liquid chromatography in which the stationary phase
is an ion exchange resin which may be cation exchange resin or
anion exchange resin.
It can be used for almost any kind of charged molecule
including large proteins, small nucleotides and amino acids.
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4. Principle
Reversible exchange of ions between ions present in solution and ion
exchange resin.
Cation exchange chromatography:
Positively charged proteins are reversibly adsorbed to immobilized
negatively charged beads/polymers.
Anion exchange chromatography:
Negatively charged proteins are reversibly adsorbed to immobilized
positively charged beads/polymers.
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5. Animated view
• Cation exchange:
• Anion exchange:
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6. Types of ion exchange resin on basis of chemical nature
Types Functional group Examples PH value
Strong acid cation
exchanger
sulfonic acid Methylsulphonate
–CH2CH2SO3–
1 to 14
Weak acid cation
exchanger
Carboxylic acid Carboxymethyl
–CH2COO–
5 to 14
strong base anion
exchanger
Quaternary amine –CH2CH2N(CH2CH3)3
+ 0 to 12
Weak base anion
exchanger
Amine Diethylaminoethyl(DEAE)
–CH2CH2NH(CH2CH3)2
+
0 to 9
Cation exchange resin: It exchanges cations.
𝑆𝑜𝑙𝑖𝑑 − 𝐻+ + 𝑀+ → 𝑆𝑜𝑙𝑖𝑑 − 𝑀+ + 𝐻+
Anion exchange resin: It exchanges negatively charged ions such as
chloride etc.
𝑆𝑜𝑙𝑖𝑑 − 𝑂𝐻− + 𝐴− → 𝑆𝑜𝑙𝑖𝑑 − 𝐴− + 𝑂𝐻−
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7. Anion exchange
Cation exchange - in
softening of water
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8. Types of ion exchange resin on basis of source
Natural resin:
 Cation exchange resin: Zeolites, clay
 Anion exchange resin: Dolomite
Synthetic resin:
 Organic and inorganic resin
 Epoxy resins
 Acetal resin
 solvent impregnated resins
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9. Types of ion exchange resin on basis of Structure
A. Peculiar types with ion exchange film
• Peculiar size of 30-40 micrometer
film thickness
• Ion exchange efficiency is
0.01-0.1 meq/g of ion exchange resin.
B. Porous resin coated with exchange beads
• Partical size of 5-10 micrometer
• Ion exchange efficiency is
0.5-2 meq/g of ion exchange resin.
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10. Types of ion exchange resin on basis of Structure
C. Macroreticular resin bead:
• Not highly efficient
• Very low exchange capacity
D. Surface sulphonated and bounded
electrosctatically with anion exchange:
• Less efficient and low exchange capacity
• Ion exchange capacity is 0.02meq/g
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11. Ion exchange resin should have following requirements:
• It must be chemically stable
• Insoluble in common solvents
• It should have a sufficient degree of cross linking
• The swollen resin must be denser than water
• It must contain sufficient no. of ion exchange groups
• A resin of known weight should be used
• Analytical grade resins are prefered as they are carefully sized and
washed.
Selection of a resin
 If a protein is most stable below its pH, a cation exchanger should be
used
 If a protein is most stable above its pH, an anion exchanger should be
used
 If stability of the protein is known to be good over a wider pH range
then either type of ion exchanger can be used 11

12. Practical requirements of ion exchange chromatography
1. Column material and Dimentions
• Material: Glass (for laboratories), high quality stainless steel or
polymers(Industries)
• Dimentions: Length diameter ratio of 20:1 to 100:1
2. Types of ion exchange resin and physical resin
• Types: Cation and anion
• Nature of ions to be seperated: Weak or strong
• Efficiency of resin: Measured by ion exchangge capacity
• Particle size of resin: 50 to 100 mesh 0r 100 to 200 mesh
• Structural types of resin: Porous, peculiar atc
• Amount of cross linking agent present which decides swelling of
resin 12

13. Practical requirements of ion exchange chromatography
3. Stationary Phase
• It is composed of two structural elements; Charged groups involved
in ion exchange and matrix on which charged groups are fixed.
• Some matrix are; Cellulose, sillia, polyacrylamide, acrylate co-
polymer, coated sillica
4. Mobile phase
• Generally, eluent which consists of aqueous solution of suitable
salt or mixture of salt with small percentage of organic compounds
is used in which all ionic compounds are dissolved.
• Some eluents used in ion exchange chromatography are EDTA,
polyols, glycerol, glucose, detergents, lipids, organic solvents, urea
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14. Practical requirements of ion exchange chromatography
5.Buffers
• In IEX, PH value is an important parameter and can be controlled by
means of buffer.
• For cation exchange; Citric acid, lactic acid, acetic acid, formic acid
• For anion exchange; Piperazine, N-methyl piperzine,
triethanolamine, Ethanolamine
6. Sample preparation
• For sample preparation, sample must be soluble in eluent and
should ideally dissolve in mobile phase itself
• To protect column from possible damage, sample must be filtered
before use to remove particulates.
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15. Practical requirements of ion exchange chromatography
7. Packing of column
• Wet packing method is th ideal technique of column packing.
• Slurry is prepared be mixing silica(10-20g) and least polar solvent in
a beaker and poured in the column.
• When the packing is complete, the eluent is allowed to pass
through colum for certein time.
8. Development of chromatogram and elution
• After introduction of sample, development of the chromatogram
is done by using different mobile phases.
• There are two elution techniques; isocratic and gradient elution
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16. Practical requirements of ion exchange chromatography
9. Analysis of elute
Different fractions collected with respect to volume or time is analysed
for their contents by several methods.
• Spectrophotometric method
• Polarographic method
• Conductometric method
• Radiochemical method
10. Regeneration of ion exchange resin
• It refers to replacement of exchangeable cations or anions present
in the original resin
• Regeneration of cation exchange resin is done by charging the
column with strong acid like HCl.
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17. Steps in ion exchange chromatography
 An IEX medium comprises a matrix of spherical particles
substituted with ionic groups.
 The matrix is usually porous to give a high internal surface area.
The medium is packed into a column to form a packed bed.
 The bed is then equilibrated
There are four steps:
• Equilibrium
• Sample application and wash
• Elution
• Regeneration
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18. 1. Equilibrium:
• The first step is the equilibration of the stationary phase to the
desired start conditions.
• When equilibrium is reached, all stationary phase charged groups
are bound with exchangeable counter ions, , such as chloride or
sodium.
• The pH and ionic strength of the start buffer are selected to ensure
that, when sample is loaded, proteins of interest bind to the medium
and as many impurities as possible do not bind.
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19. 2. Sample application and wash
• The goal in this step is to bind the target molecule(s) and wash out
all unbound material.
• The sample buffer should have the same pH and ionic strength as
the start buffer in order to bind all charged target proteins.
• Oppositely charged proteins bind to ionic groups of the IEX
medium, becoming concentrated on the column.
• Uncharged proteins, or those with the same charge as the ionic
group, pass through the column at the same speed as the flow of
buffer, eluting during or just after sample application, depending on
the total volume of sample loaded.
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20. 3. Elution
• After sample application and washing, conditions are altered in
order to elute the bound proteins.
• Most frequently, proteins are eluted by increasing the ionic strength
of the buffer or, occasionally, by changing the pH. As ionic strength
increases the salt ions compete with the bound components for
charges on the surface of the medium and one or more of the bound
species begin to elute and move down the column.
• The proteins with the lowest net charge at the selected pH will be
the first ones eluted from the column as ionic strength increases.
• The proteins with the highest charge at a certain pH will be most
strongly retained and will be eluted last.
• The higher the net charge of the protein, the higher the ionic
strength that is needed for elution.
• By controlling changes in ionic strength proteins are eluted
differently in a purified, concentrated form. 20

21. 3. Elution
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22. 4. Regeneration
• A final wash with high ionic strength buffer regenerates the column
and removes any molecules still bound. This ensures that the full
capacity of the stationary phase is available for the next run.
• The column is then re-equilibrated in start buffer before starting the
next run.
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23. Ion exchange techniques
Two techniques are generally used to bring the solution in contact with
ion exchange resins. They are:
1. Batch method
2. Column method
1. Batch method
• This method involves single step equilibrium.
• The resin and the solution are mixed in a vessel until equilibrium
is attained.
• The solution which is obtained filtered.
• In single step equilibrium of this type, only a single portion of the
exchange capacity of the resin is utilized.
• This method is used for softening of water and the production of
deionized or demineralised water
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24. Ion exchange techniques
 The apparatus used in the column method consist of a glass
column fitted with a glass wool plug or a sintered glass disc at
the lower end.
 An ordinary burette can also be used.
 The resin used should have small particle size and the diameter
of the resin should be less than one tenth of the column.
 The slurry is slowly poured into the column containing some
water.
 The resin is allowed to settle and then excess water is drained off
2. Column Method
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25. 25

26. Chromatographic parameters
1. Rate of ion exchange process:
Its depends on the rate of diffusion which is the slowest state on ion
exchange process.
2. Flow rate:
Due to difference in rate of exchange and the fact that they may
vary significantly for different kinds of seperation, flow rates are to
be controlled (0.5-5 ml/min)
3. Mechanical strength:
Polystyrene bead would have little mechanical strength and upon
adding functional group such as
sulphonic acid to ploymer, solubility is enchance greatly
If polymer is cross linked by incorporation of divinyl benzene,
mechanical strength is imparted to resin.
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27. Chromatographic parameters
3. Swelling:
Swelling is due to the tendency of particles to hydrate and
electrostatic repulsion of fixed with like charges.
Swelling is continued until an equilibrium is reached between the
osmotic pressure in the system and opposing elastic force of flexible
hydrocarbon chains.
4. Particle size:
Large surface area and small particle size will increase the rate of ion
exchange process
5. Porosity:
High porosity offers a large surface area covered by charged groups
ans so provides a high binding capacity.
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28. Chromatographic parameters
7. Selectivity:
• The ion exchange in solution is a selective process.
• As absolute conc. Of solution decreases, polyvalent are absorbed
and at higher concentration, monovalent are adsorbed
8. Total capacity:
Total capacity of resin is determined by taking a weighed sample of
resin, placing it in a colum and passing through a solution of KCl
through the column in excess.
9. Exchange capacity:
It depends on the wuality of the ions extracted from water by ine
gram of air dry ion exchanger.
10. Crosslinking:
• As crosslinking decreases, resin swelling increases.
• Divinyl is the most commonly employed crosslinking agent. 28

29. Factors affecting ion exchange chromatography
Three major factors are affecting ion exchange selectivity in IEC.
• Cross linking and swelling are important factors, when more cross
linking agent is present, the resin becomes more rigid and swells
less (has small pore size). This makes separations of ions of
different sizes more difficult as they can not pass through the pores
present and it becomes selective to ions of different (smaller) sizes.
• The nature of resin whether cationic or anionic exchanger, which
determines strongly its selectivity. Cationic resin is selective for
cations and vice versa.
• The resin capacity (number of me-equivalents of replaceable ions
per gram of dry resin) is important.
1. Nature and properties of ion exchange resins
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30. 2.Nature of exchanging ions in the sample
a. Valence of ions:
• At low concentrations, the extent of exchanges increases with
increase in valence; Ions with higher charge is more selective
𝐶𝑒4+ < 𝐴𝑙3+ > 𝐵𝑎2+ > 𝑇𝑖+
b. Size of ions:
• For similarly charged ions; the exchange selectivity increases with
decrease in the size of hydrated ions;
Li+ < H+ < Na+ < K+< Rb+ < Cs+< Ag+
c. Polarizability:
Highly polarizable ions are more selective. Exchange is preferred
for greater polarizable ions, as;
d. Concentration of solutions:
In dilute solutions poly-valent ions are generally absorbed
preferentially.
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31. 3.Nature of mobile phase and pH
Exchange constant or selectivity coefficient (K)
• The presence of other ions that compete with the sample for
binding to the ion exchanger (using of electrolyte).
• The pH of the solution which influences the net charge of the
sample (as in case of amino acids).
𝑆𝑜𝑙𝑖𝑑 − 𝐻+ + 𝑀+ ⇋ 𝑆𝑜𝑙𝑖𝑑 − 𝑀+ + 𝐻+
For above equilibrium
𝐾 =
𝑆𝑜𝑙𝑖𝑑 − 𝑀+ 𝐻+
𝑆𝑜𝑙𝑖𝑑 − 𝐻+ 𝑀+
𝑆𝑜𝑙𝑖𝑑 − 𝑂𝐻− + 𝐴− ⇋ 𝑆𝑜𝑙𝑖𝑑 − 𝐴− + 𝑂𝐻−
For above equilibrium
𝐾 =
𝑆𝑜𝑙𝑖𝑑 − 𝐴− 𝑂𝐻−
𝑆𝑜𝑙𝑖𝑑 − 𝑂𝐻− 𝐴−
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32. Exchange constant or selectivity coefficient (K)
32

33. Applications of ion exchange chromatography
IEC is utilized in numerous industrial and research settings including
a test for authentic tequila and for environmental analyses such as the
determination of anions (phosphate, chloride, etc) in surface waters.
 It is used in the treatment of water for drinking, use
(commercial, industrial, and residential), and wastewater
treatment. Ion exchangers can soften the water, deionize it, and
even be used in desalination.
 Preparation of various acids, bases, salts, and solutions is also
aided by ion exchange.
 The recovery of valuable metals is also possible with resins.
 Industrial drying of treatment of gases is accomplished often with
ion exchange.
 The food industry uses ion exchange in a variety of ways, ranging
from wine-making to sugar manufacture.
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34. Applications of ion exchange chromatography
 In the medical world, it is used for development and preparation of
key drugs and antibiotics, such as streptomycin and quinine, for
treatments of ulcers, TB, kidneys, and much more.
 Ion exchange is used to prevent coagulation in blood stores and in
dextrose, as well.
 An ion exchange is also useful in death, as it plays a role in the
treatment of formaldehyde.
 In soil science, cation exchange capacity is the ion exchange
capacity of soil . Soils can be considered as natural weak cation
exchangers.
 A very important case is the PUREX process (plutonium-uranium
extraction process), which is used to separate the plutonium and
the uranium from the spent fuel products from a nuclear reactor.
 In planar waveguide manufacturing, ion exchange is used to create
the guiding layer of higher index of refraction.
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35. Applications of ion exchange chromatography
• An important use of ion-exchange chromatography is in the
routine analysis of amino acid mixtures.
• The 20 principal amino acids from blood serum or from the
hydrolysis of proteins are separated and used in clinical diagnosis.
• In the analysis of products of hydrolysis of nucleic acids. In this
way, information is gained about the structure of these molecules
and how it relates to their biological function as carriers of
hereditary information.
• Chelating resins are used to collect trace metals from seawater.
• To analyze lunar rocks and rare trace elements on Earth.
• Removal of interfering ions For estimation of 𝐶𝑎2+, 𝐵𝑎2+ions by
the oxalate or sulphate method in quantitative analysis, phosphate
ions are found to interfere and can be removed by passing the
solution through sulphuric acid.
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36. Advantages of ion exchange chromatography
• Has highest resolving power
• Has highest loading capacity
• Widespread applicability (almost universal)
• Most frequent chromatographic technique for protein purification
• Used in approximately 75% of all purifications
• It is one of the most efficient methods for the separation of charged
particles.
• Can be used for almost any kind of charged molecule including
large proteins, small nucleotides and amino acids.
• Ion exchange is used for both analytical and preparative purposes in
the laboratory, the analytical uses being the more common.
• Inorganic ions also can be separated by ion-exchange
chromatograph.y
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37. Thank you
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