Ion exchange chromatography uses ion exchange resins to separate ionic compounds. The document discusses the principles and process of ion exchange chromatography. It involves an ionic compound binding electrostatically to functional groups on a solid resin through ion exchange. The ions can then be separated and eluted by changing the mobile phase solution. The document provides details on the types of ion exchange resins, factors affecting ion separation, and applications such as producing deionized water and separating amino acids and lanthanides.

1. ION EXCHANGE CHROMATOGRAPHY
H+
H+ Na+
R
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2. ION EXCHANGE CHROMATOGRAPHY
H+
OH-
Cl-
R
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3. ION EXCHANGE
CHROMATOGRAPHY
H+
Stationary
Phase ( Solid)
Ion exchange Resin
Mobile phase
Liquid
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4. Ion Exchange
Ion exchange is an adsorption
phenomenon where the mechanism of
adsorption is electrostatic. Electrostatic
forces hold ions to charged functional
groups on the surface of the ion
exchange resin. The adsorbed ions
replace ions that are on the resin surface
on a 1:1 charge basis. For example:
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5. H+
Na+
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6. • The first ion exchangers were synthetic resins
designed for Applications such as
demineralization, water treatment, and recovery
of ions from wastes. The first ion exchangers
designed for use with biological substances were
the cellulose ion exchangers developed by
Peterson and Sober.
• Ion exchangers based on dextrin (Sephadex), followed
by those based on agarose (Sepharose CL-6B) and
cross-linked cellulose (DEAE Sephacel) were the first
ion exchange matrices to combine a spherical form
with high porosity, leading to improved flow properties
and high capacities for macromolecules
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7. What are ion
exchange resins ?
Polymeric resins are made in 3-D
networks by cross-linking hydrocarbon
chains. The resulting resin is insoluble,
inert and relatively rigid. Ionic
functional groups are attached to this
framework
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8. INTEGRAL PART
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9. Ion exchange resins
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10. CHARACTERISTICS
OF
ION EXCHANGE
RESINS
It should be insoluble
in aqueous medium.
Should be denser than
water
Should have lose
porous polymeric
structure.
It should be inert in
nature
Should have large
exchangeable sites
Should have high
degree of cross linking.
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11. CLASSIFICATON
OF
ION EXCHANGE
RESINS
CATION EXCHANGE
RESINS
STRONGLY
ACIDIC CATION
EXCHANGE RESIN
WEAKLY
ACIDIC CATION
EXCHANGE
RESIN
ANION EXCHANGE
RESINS
STRONGLY
BASIC ANION
EXCHANGE RESIN
WEAKLY
BASIC ANION
EXCHANGE RESIN
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12. STRONGLY
ACIDIC
CATION
EXCHANGE
RESIN
R SO3H
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13. WEAKLY
ACIDIC
CATION
EXCHANGE
RESIN
R COOH
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14. STRONGLY
BASIC
ANION
EXCHANGE
RESIN
R Cl-
R=QAS
Ex. R-
(NCH3)3+ Cl--
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15. WEAKLY
BASIC
ANION
EXCHANGE
RESIN
R OH-
R= Primary
amino group
or secondary
amino group
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17. Working Principle
•The basic process of chromatography using ion
exchange can be represented in following steps:
1. Equilibrium
2. Sample application
3. Elution
4. Regeneration

23. 3) Stationary phase:
● It is composed of two structural elements; the charged groups which are involved inthe
exchanger process and the matrix on which the charged groups arefixed.
● Sever matrix materials are- Cellulose,Silica,Polyacrylamide,Acrylate co-polymer,Coated silica
4) Mobile phase:
● Generally, eluents which consist of an aqueous solution of a suitable salt or mixtures of thesalt
with a small percentage of an organic solvent are used in which most of the ionic compounds
are dissolved.
● There are some eluent additives which have been used in ion exchange chromatography-
EDTA,Polyols,Glycerol,Glucose,Detergents,Lipids,Organic solvents,Urea .
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, Formicacid
● For Anion exchange chromatography-Piperazine,N-Methyl
piperazine,Triethanolamine,Ethanolamine
9

24. 5)Sample Preparation:
● For sample preparation,the sample must be soluble in eluent & should ideally be dissolved in the
mobile phase itself.
● To protect the column from possible damage, samples are filtered before use to remove
particulates.
6)Packing of the column:
● Wet packing method is the ideal technique of column packing in ion exchange chromatography.
● Slurry is prepared by mixing silica(10-20g) and least polar solvent in a beaker & poured inthe
column.
● When the packing is complete, the eluent is allowed to pass through column for certaintime.
7)Devolepment of the chromatogram & elution:
● After introduction of the sample, devolepment of the chromatogram is done by usingdifferent
mobile phases.
● There are two elution techniques-Isocratic & Gradient elution. 10

25. 7)Analysis of the 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
8)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 hydroxideor
potassium hydroxide. 11

26. INSTRUMENTATION
(a) BATCH METHOD
● This involves a single step equilibrium.
● The resin & the solution are mixed in vessel until the equilibrium is attained & the solution isthen
filtered off.
● The batch method is used for softening of water & production of demineralisedwater.
● Softening of water involves an exchange of calcium & magnesium ions,which cause hardnessby
sodium ions.The sodium form of sulphonic acid is generallyused.
𝟐𝑹𝑺𝑶₃−𝑵𝒂+ +𝑪𝒂𝟐+ (𝑹𝑺𝑶𝟑)₂−𝑪𝒂𝟐+ +𝟐𝑵𝒂+
● Demineralised water is prepared by treating water with a cation exchanger in the acid or hydrogen
forms.
𝑹𝑺𝑶₃−𝑯+ +𝑴+ 𝑹𝑺𝑶₃−𝑴+ +𝑯+
● The water is then treated with an anion exchanger in the basic or hydroxideform.
𝑹+𝑶𝑯− +𝑪𝒍− 𝑹+𝑪𝒍− +𝑶𝑯− 12
techniques

27. 13

28. (b) 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.
● The flow of water is stopped and the resin is allowed to settle.
● The excess water is then drained off.
14
● 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.

30. 1 • The charge on the ion.
2 • The size on the ion
• .
3 • The concentration of the ion
4
• Use of complexing agent and ability to form
complexes
5 • Nature of the resin.
6 • Effect of pH.
Factors affecting separation of Ions:
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31. The charge on the
ion
Attraction of the resin for an ion
increases with increase in the charge
carried by the ion. The preference of
the resin with the ion can be follow
the order.
Ex. Th4+ >Al3+ >Ca2+>Na+
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32. The size of the ion
The size of the bare ion is not considered here
because ions get hydrated when they are in the
aqueous medium.
The ions having small size get hydrate more and
have less affinity with resin and vice versa i.e.
The ions having large size get hydrate less and
have more affinity with the resin.
Ex. Li+ <H+ <Na+< NH4+ < K+<Rb+
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33. Resin
Hydrated
Size of an
ion
Small
size
ion
Large
size ion
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34. The concentration
of the ion:
Higher the concentration of the ion
(same ion) in the solution higher is the
affinity of the ion with the resin.
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35. The
concentration
of the ion
Affinity of
Resin
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36. Use of Complexing
agent and ability to
form complexes :
The ion having ability to form
complexes its size will increases and
its affinity with the resin is decreases
and vice versa.
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37. Nature of the
resin.
Resin having spherical shape,
high degree of cross linking,
strength of functional group
decides its ability for the
exchange of ions.
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38. Effect of pH :
The pH can affect on separation by several ways, The ioniz--
-ation of the resin in solution depends on the pH especially if
the functional group is weakly acidic or basic. The strength
of acids or bases, the hydrolysis of the salts, the ionization of
the functional group of the resin, all are strongly dependent
on pH of the solution.
For better ion exchange, weakly acidic anion exchanger
has to used in alkaline medium.
Similarly weakly basic anion exchange has to use in
acidic medium
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39. ION EXCHANGE CAPACITY
Efficiency of ion exchange process
depends upon exchange capacity of resin.
The number of mill equivalent ion
exchange by one gram of dry resin is called
as ion exchange capacity
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40. DETERMINATION OF CATION
EXCHANGE CAPACITY
One gram of cation exchange resin is soaked in HCl acid to
convert the resin in H+ form. It is then placed in water. The
glass tube like that of burette is packed with the resin. 10 ml
of 0.5N solution of Na2SO4 is placed on the resin column.
The elution is carried out using distilled water. The eluate is
collected in conical flask and then titrated with 0.1N NaOH
solution using phenolphthalein indicator.
Volume of NaoH required for the titration is found out
and cation exchange capacity is determined by using
following formula. =
V x N
W
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41. DETERMINATION OF ANION
EXCHANGE CAPACITY
One gram of anion exchange resin is soaked in HCl acid to
convert the resin in Cl- form. It is then placed in water. The
glass tube like that of burette is packed with the resin. 10 ml
of 0.5N solution of NaCl is placed on the resin column. The
elution is carried out using distilled water. The eluate is
collected in conical flask and then titrated with 0.1N AgNO3
solution using potassium chromate as indicator.
Volume of AgNO3 required for the titration is found out
and Anion exchange capacity is determined by using
following formula. =
V x N
W
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42. EXPERIMENTAL TECHNIQUE
Ion Exchange Resin
Cotton Plug Or glass
wool plug
Water level
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43. Terms involved
• The process of removing
adsorbed ions is known
as elution.
Elution
• The solution used for
elution is called as eluent
.
Eluent
• The solution resulting
from the elution is called
as eluate.
Eluate
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44. APPLICATION OF
ION EXCHANGE
CHROMATOGRAPHY
Preparation of deminerlised
water or deionised water.
Separation of Lanthanides.
Separation of amino acids.
Determination of
concentration of trace
constituent.
Separation of similar ions.
Separation of interfering
cations and anions.
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45. 2/9/2022 45

46. Preparation of deionsied Water
Water from which all cations and anions are removed is called as Deminerlised
water or deionised water.
The process involves two steps.
a) In the first step water is passed through the cation exchange resin column (H+
form). While passing through the column all cations such as Na+, Mg2+ are
exchanged for H+ ion .
R-SO3-H+ + M+ --------- RSO3- M+
-----------
b) In second step, water coming out from first column is allowed to pass through an
anion exchanger in basic form (OH-) In this anions like Cl- ,SO42- are exchanged
for OH-.
R- OH- + Cl-+ --------- R--Cl- + OH-.
-----------
H+ ions combine with OH- to give unionized water.
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47. R-H+
Cation
Exchange
Resin
R-OH-
Anion Exchange
Resin
H+
OH-
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48. 2) Separation of Lanthanides:-
La
Ce
Pr
Nd
Pm
Sm
Eu
Gd
Tb
Dy
Ho
Er
Tm
Yb
Lu
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49. 2) Separation of Lanthanides:-
57 La
58 Ce
59 Pr
60 Nd
61 Pm
62 Sm
63 Eu
64 Gd
65 Tb
66 Dy
67 Ho
68 Er
69 Tm
70 Yb
71 Lu
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Ionic Radius
Hydrated size
of ion
Affinity of an
ion with
Resin
Separation of
Lanthanides
71 Lu
70 Yb
69 Tm
68 Er
67 Ho
66 Dy
65 Tb
64 Gd
63 Eu
62 Sm
61 Pm
60 Nd
59 Pr
58 Ce
57 La
Lanthanide
Contraction
La is bigger in size
so less hydration
more association with resin
so come out last

50. 2) Separation of Lanthanides:-
In this column is packed with cation exchange resin of type R-H. When
solution containing mixture of lanthanides is poured on the top of the hen
following equilibrium takes place,
3R-H + Ln3+ ---------------- R3 ____Ln 3+ 3H+
<--------------
In case of lanthanides the ionic radii of tripositive ions decreases with
increase in atomic number i.e. from lanthanum to lutecium.
Hydration of ion increases with decrease in ionic size and therefore hydrated
size of lanthnone ion increases with increase in atomic number.
Lanthnone ion having small hydrated size( La) will strongly adsorb on the
resin and vise versa (Lu). Hence lanthnone ion held by resin decreases from
La to Lu.
When HCL solution is passed as mobile phase separation of lanthanides takes
place in reverse order of atomic number. Lutecium ion will separate out first
where as Lanthanum ion separate at last.
For better separation elution is carried out using ammonium Citrate buffer at pH
2.8 -3.4.
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51. Separation of amino acids:-
Chromatographic column is packed with
cation exchange resin. Solution of metal
ions like Cu2+,Cd2+ are exchanged on the
column. When amino acid mixture passed
on the column it forms complexes with
varying stability. Most stable complex will
elutes first and least stable will elutes at
last.
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52. Determination of concentration
of trace constituent.
Trace amount substance present in large volume
can be found out.
Ex. If trace amount of Ag+ ion is present in large
volume of water .In this Column is packed with
Cation exchange resin. Then solution is passed.
Ag+ ion exchange with cation. Silver is then
eluted by using small amount of eluent.
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53. Separation of similar ions
Ex. Similar ions like Cl-,Br- and I- can be separated using
ion exchange chromatography. Column is packed with
anion exchange resin like R-OH- .When solution
containing halides passed over the column these anions
will adsorb at various extent. When elution is carried out
using 0.3M NaNO3 Solution Cl-will elutes out first.
When concentration NaNO3 increases to 0.6M After
elution Br - ion eluted similarly I- ion eluted at last using
0.9M NaNO3.
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54. Separation of interfering cations
and anion
Interfering ion can be removed by ion exchange
chromatography. Ex. In the estimation of Ca2+ and
Mg2+ion by sulphate method, Phosphate ion
interfere in the estimation. This Phosphate ion can
be removed by passing solution through cation
exchanger, Ca2+ and Mg2+ions get exchanged for
H+ ion. While phosphate ion s pass through
exchanger. Ca2+ and Mg2+ions held by resin are
eluted and then estimated.
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