simple and basic

1. ION-EXCHANGE
CHROMATOGRAPHY
Submitted by:
Rishikesh A. Tiwari
M.Pharm.(Pharmaceutical Quality
Assurance) –Semester 1
Guided By:
Ms. Bhumika Maheriya
Assistant Professor
GTU-GSP
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2. CONTENTS
› HISTORY
› DEFINITION
› PRINCIPLE
› CLASSIFICATION OF RESINS
› APPARATUS OF ION EXCHANGE CHROMATOGRAPHY
› INSTRUMENTATION
› ION EXCHANGE TECHNIQUES
› CHROMATOGRAPHIC PARAMETERS
› FACTORS AFFECTING RESOLUTION
› APPLICATIONS OF ION EXCHANGE CHROMATOGRAPHY
› REFERENCE 2

3. History of IE
Chromatography
› 1850 - H. Thompson and J.T way who proved that soil
can remove potassium or ammonium salts from water
with release of equivalent amount of calcium salt
› Natural and synthetic inorganic cation exchangers were
used for softening hard water after the work of Gans in
1913.
› 1927 -Zeolite mineral columns were used to remove
interfering calcium and magnesium ions from solution to
determine sulfate content of water 3

4. › Modern ion exchange resin were first used in 1935 by
Adams and Holms.
› 1940s Modern Ion-Exchange Chromatography was
developed during the wartime Manhattan Project.
› This technique was used to separate and concentrate the
radioactive elements needed to make an atomic bomb.
› The adsorbents would latch onto charged transuranium
elements differentially eluting them.
4

5. › 1970s Hamish Small and co-workers of Dow Chemical
Company developed ion-chromatography usable for
automated analysis.
› IC uses weaker ionic resins for the stationary phase and a
neutralizing stripper column to remove background eluent
ions.
› Used for determining low concentrations of ions in water
and other environmental studies.
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6. DEFINITION
› Ion exchange chromatography is the 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.
›
6

7. Terminology
• Influent – The liquid entering the column.
• Effluent – The liquid leaving the column.
• Elution – The process by which the adsorbed ions are
removed from the column.
• Eluent – The solution used for elution.
• Eluate – The solution obtained as result of elution.
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8. PRINCIPLE
› It is based on the reversible electrostatic
interaction of ions with the separation matrix (i.e.)
The separation occurs by reversible exchange of
ions between the ions present in the solution and
those present in the ion exchange resin.
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9. 9

10. Components of IEC
› Stationary phase or Ion exchange resin fixed charged groups and
replaceable counter ions of opposite charge.
• Liquid phase contains molecules of organic or inorganic ions .
› Solutions of different charges to influence interactions between
liquid and solid phases.
• Ion exchange takes place and principle of
electro- neutrality is maintained all the time.
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11. › The ion exchange resin is an insoluble polymer
consisting of a "matrix" (Lattice or framework) that
carries fixed charges (not exchangeable) and mobile
active ions "counter ions" which are loosely attached
to the matrix.
In water, the counter-ions move more or less freely in
the framework & can be replaced by ions of the
same sign present in the surrounding solution.
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ION EXCHANGE RESIN

12. COMMON REQUIREMENT OF ION EXCHANGE
RESIN
● They are almost insoluble in water and organic solvents such
as benzene, carbon tetrachloride, ether etc.
● They are complex in nature ,i.e. in fact they are polymeric.
● They have active or counter ions that will exchange reversible
with other ions in a surrounding
solution without any substantial change in the material.
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13. PHYSICAL PROPERTIES OF RESINS
› Cross linking:
It affects swelling & strength & solubility
› Swelling:
When resin swells, polymer chain spreads apart
Polar solvents → swelling
Non-polar solvents → contraction
Swelling also affected electrolyte concentration
› Particle size and porosity
Increase in surface area & decrease in particle
size will increase the rate of ion exchange.
›
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14. › Regeneration
Cation exchange resin are regenerated by
treatment with acid, then washing with water.
Anion exchange resin are regenerated by
treatment with NaOH, then washing with water
until neutral.
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15. CLASSIFICATION OF ION EXCHANGER
1) ACCORDING TO THE SOURCE:
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16. 2) ACCORDING TO THE STRUCTURE:
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17. CLASS OF
RESIN
NATURE PH RANGE APPLICATION
Cation -
Strong
Sulfonated
polystyrene
1-14 -fractionation of caption's
-inorganic separation (lanthanides)
-peptides, amino acids, B vitamins
Cation -
Weak
Carboxylic
methacrylate
5-14 -fractionation of caption's
-biochemical separation
-organic bases, antibiotics
Anion -
Strong
Quaternary
ammonium
polystyrene
0-12
-fractionation of anions
-alkaloids, vitamins
-fatty acids
Anion -
Weak
Polyamine polystyrene
or phenol
0-9
-fractionation of anion complexes
-anions of different valency
3) ACCORDING TO THE NATURE
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18. INSTRUMENTATION OF IE CHROMATOGRAPHY
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19. 1)Column:
Glass, stainless steel or polymers which are
resistant to strong acids and alkalis.
2)Selection of ion exchange resin:
 Type of the ions to be separated – cations or anions
 Nature of the ions to be separated- strong or weak
 Efficiency of the resin: measured by ion exchange
capacity
 Particle size of the resin: 50-100 mesh or 100-200
 Structural type of the resin: porous, peculiar etc.
 Amount of cross linking agent present: which decides
swelling of the resin.
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20. 3)Packing of the column:
› Wet packing
› The resin is mixed with the mobile phase and packed in
the column uniformly
› The sample to be separated is dissolved in the mobile
phase and introduced all at once into the column
4)Application of the sample:
› After packing, sample is added to the top of the
stationary phase, use syringe or pipette
› This layer is usually topped with a small layer of sand or
with cotton or glass wool to protect the shape of the
organic layer from the velocity of newly added eluent.
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21. 5)Mobile phase
Acids, alkalis, buffers…
6)Stationary phase
› The ionic compound consisting of the cationic species
(M+) and the anionic species (B-)
› Several matrix materials are- Cellulose, Silica,
Polyacrylamide, Acrylate co-polymer, coated silica
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22. 7)BUFFERS:
› In ion exchange chromatography, PH value is an
important parameter for separation & can be controlled
by means of buffer substances.
› For Cation exchange chromatography- Citric acid, Lactic
acid, Acetic acid, Formic acid
› For Anion exchange chromatography- Piperazine,N-
Methylpiperazine,Triethanolamine,Ethanolamine
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23. 8)Elution
› Components of mixture separate & move down the
column at different rates depending upon the affinity of
the ion for ion exchanger.
› The elutes are collected at different stages
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24. 9)Analysis of the elute:
› Different fractions collected with respect to volume or
time is analyzed for their contents by several methods.
› Spectrophotometric method
› Polarographic method
› Conductometric method
› Radiochemical method
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25. 10)Regeneration of the ion exchange resin:
› Regeneration refers to the 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 hydrochloric
acid.
› Regeneration of anion exchange resin is done by using
strong alkali like sodium hydroxide or potassium
hydroxide. 25

26. ION EXCHANGE TECHNIQUE
1)BATCH METHOD
› This involves a single step equilibrium.
› The resin & the solution are mixed in vessel until the
equilibrium is attained & the solution is then filtered off.
› The batch method is used for softening of water & production
of demineralized water.
› Softening of water involves an exchange of calcium &
magnesium ions, which cause hardness by sodium ions. The
sodium form of sulphonic acid is generally used. 26

27. › Demineralized water is prepared by treating water with a
cation exchanger in the acid or hydrogen forms.
› The water is then treated with an anion exchanger in the
basic or hydroxide form.
27

28. 2)COLUMN METHOD:
› The apparatus used in the column method, consist of a
glass column fitted with a glass wool plug or a sintered
glass disc at a lower end.
› A slurry of resin is made in distilled water and any fine
particles are removed by decantation.
› The slurry is then slowly poured into the column.
› To ensure that no air bubbles remain in the column and
that the resin is uniformly distributed, the column is
backwashed with distilled water. 28

29. › The flow of water is stopped and the resin is allowed to
settle.
› The excess water is then drained off.
› The level of water must never be allowed to fall below
that of the surface of the resin as otherwise the resin may
dry up and channels may be formed in the resin bead.
2.1)Frontal Analysis:
› In Frontal analysis, an incomplete separation of ion is
obtained.
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30. 2.2)Displacement Development:
› Displacement development of the column is
accomplished by means of a substance which has a very
strong affinity for the exchanger.
2.3)Elution development:
› When the elution development is performed, the
components of a mixture separate and move down the
column individually at different rates depending on the
affinity of the ion for exchanger.
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31. 31

32. CHROMATOGRAPHIC PARAMETERS
1)Rate of ion exchange process:
The rate of ion exchange process depends on the rate of diffusion
which is the slowest state on ion exchange process.
2)Flow rate:
Due to differences in rates of exchange and the fact that they may
vary significantly for different kinds of separation, flow rates are to be
controlled (0.5-5 ml/min).
3)Mechanical strength:
If the polymer is crosslinked by the incorporation of divinyl benzene,
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33. 4)Swelling:
● Swelling is due to the tendency of particles to hydrate
and electrostatic repulsion of fixed ions with like charges
● Swelling is continued until an equilibrium is reached
between the osmotic pressure in the system and the
opposing elastic force of the flexible hydrocarbon chains.
5)Particle size:
● Large surface area and small particles will increase the
rate of ion exchange process.
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34. 6)Porosity:
● High porosity offers a large surface area covered by charged
groups and so provides a high binding capacity.
7)Selectivity:
● The ion exchange in solutions is a selective process.
● At the absolute concentration of the solution decreases,
polyvalent ions are adsorbed better than monovalent ions, while at
higher concentrations, monovalent ion is adsorbed.
8)Total capacity:
● Total capacity of a resin is determined by taking a weighed sample
of resin, placing it in a column and passing through a solution of Kcl
through the column in excess.
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35. 9)Exchange capacity:
● It depends on the quality of the ions extracted from water by one gram
of air dry ion exchanger.
10)Crosslinking:
● As the crosslinking in the resin decreases,the resin swelling increases.
● Divinyl benzene is the most commonly employed crosslinking agent
and imparts strength to the polymer by joining the chains together at
various positions.
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36. FACTORS AFFECTING RESOLUTION
› At ordinary temperature, the extent of exchange increases
with increasing valency of the exchanging ion, provided
the concentration is low.
› Under similar conditions & constant valency, for univalent
ions, the extent of exchange increases with decrease in
size of hydrated cation.
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37. › For strongly basic anion exchange resin, the behaviors of
univalent anions is similar to univalent cations.
› When a cation in solution is being exchanged for an ion
of different valency, the relative affinity of the ion having
higher valency increases in direct proportions to the
dilutions.
› Nature of exchanging ions
› Nature of ion exchange resin
› Chemical variables
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38. › Physical variables
› Ion exchange constant
› Surface area
› Temperature
› Composition of cation exchange resin
› Length of the column
› Ionic strength
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39. APPLICATIONS OF ION EXCHANGE
CHROMATOGRAPHY
› Total content of cation in a solution
› Concentration of traces of an electrolyte
› Conversion of salts to acids or bases
› Separation of amphoteric metals from non-amphoteric
metals
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40. › Separation of metals, alloys & high alloy steels
› Analysis of natural & industrial water
› Separation of complex mixtures of biochemical
compounds
› Production of analytical concentrates
› Radiochemistry
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41. Reference
1) www.harvardapparaturs.com, guide to ion exchange
chromatography; December 5, 2019.
2) Chatwal GR, Anand SK; Instrumental analysis of chemical analysis,
Pg. 2.662-2.672.
3) Sharma BK; Instrumental methods of chemical analysis, Pg. 123 -
150.
4)http://www.gelifesciences.com/file_source/GELS/Service%20and
%20Support/Documents%20and
%20Downloads/Handbooks/pdfs/Ion%20Exchange%20Chromatogr
aphy.pdf; December 5, 2019.
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