Ion exchange chromatography

IonIon ExchangeExchange
ChromatographyChromatography
MAHENDRA G S
M.pharm

TerminologyTerminology
• Influent – The liquid entering the column.
• Effluent – The liquid living 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.

History of IE ChromatographyHistory 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 (sodium aluminum silicates) mineral columns
were used to remove interfering calcium and magnesium
ions from solution to determine sulfate content of water.
• Modern ion exchange resin were first used in 1935 by
Adams and Holms.

History of IE ChromatographyHistory of IE Chromatography
• 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.
• 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.

Chromatograhy – Mechanism of SeparationChromatograhy – Mechanism of Separation
Adsorption Partition Ion exchange

Ion-ExchangeIon-Exchange
• Usually employed with HPLC
• Ions are charged molecules
- cation positively charged ion
- anion negatively charged ion
• These ions do not separate smoothly under the
traditional methods of the liquid and mobile phases of
chromatography.
• Alteration of either the mobile phase or stationary
phase are required.
- mobile phase suppresses the ionic nature of the
analyte.
- stationary phase incorporate ions of the opposite
charge to attract and retain analyte.

DefinitionDefinition
Ion exchange may be defined as the reversible
reaction in which free mobile ions of a solid (called
ion exchange) are exchanged for different ions of
similar charge present in solution.
OR
Separation in Ion-exchange Chromatography is
based on the competition of different ionic
compounds of the sample for the active sites on the
ion-exchange resin (column-packing).
SO3
-
Na
+

Basis for Molecular SeparationBasis for Molecular Separation
• Ion ExchangeCharge

Components of IECComponents of IEC
• Stationary phase or Ion exchange resin
o fixed charged groups and replaceable
counter ions of opposite charge.
o counter ion exchange takes place
• 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.

Solid Matrix ExchangersSolid Matrix Exchangers
• 1. Cation exchanger:
o Matrix negative charge
o Exchanges cations (Counter ions are Cations)
• 2. Anion exchanger:
o Matrix positive charge
o Exchanges anions (Counter ions are Anions )

Ion exchange ResinsIon exchange Resins
• Crossed linked polystyrene
• Formed by the Copolymerization of Styrene and Divinyl
benzene.
• DVB= 8% generally used (4 -12%)
• Four types:
o Strong Cation Exchange Resin
o Strong Anion Exchange Resin
o Weak Cation Exchange Resin
o Weak Anion Exchange Resin

Process by which ions of an electrolyte solution are
brought into contact with an ion exchange resin.
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.
The "matrix" (framework) of a "cation exchanger" is
considered as a crystalline non-ionized "polyanion" &
the matrix of an "anion exchanger" as a non-ionized

PrinciplePrinciple
Reversible exchange of ions between the ions present in
sample and ion exchange resin usually carried out in
columns consisting of ion exchange resin.

How do you get thoseHow do you get those
columns to workcolumns to work
• There is a glass column
coated with a resin
polymer
• The resin is either
positively charged (an
acid) or negatively
charged (a base)
• An analyte will have
ions opposite of the
resins charge eluting off
the ion of interest

Stepwise elution for ion exchangeStepwise elution for ion exchange

Ion ExchangeIon Exchange
Low salt High salt

Fundamental requirements of a resinFundamental requirements of a resin
• A useful resin must be sufficiently hydrophilic to
permit diffusion of ions through the structure at a
finite and usable rate.
• It must be sufficiently cross linked to have only a
negligible solubility.
• It must be chemically stable and must contain
sufficient number of accessible ion exchange
groups.
• Reversibility and no permanent change in structure.
• When swollen it must be denser then water.

Common properties of ion exchangersCommon properties of ion exchangers
• Ion exchangers are complex, porous and polymeric
in nature.
• It is almost insoluble in water and organic solvents
like benzene, ether.
• They posses active or counter ions that are easily
exchangeable reversibly with other ions in
surrounding solution without any change in
material.
• Granular resins swell in water to give a gel structure.
Swelling is directly proportional to the percentage
of cross-linking. Highly cross-linked resins are brittle,
harder, complex and porous.
• Divinyl benzene prevents resin from swelling
indefinitely.

Types of Ion ExchangersTypes of Ion Exchangers
 Currently most of the common ion exchange
products are built on the styrene divinyl backbone
because of their stability.
 These polymers carry an electric charge that is
exactly neutralised by the charges on the counter
ions.
 These active ions are cations in cation exchanger
and anions in anion exchanger.
 Thus, an anion exchanger contains polymeric
cations and active anions while cation exchanger
consists of polymeric anions and active cations.

Cation ExchangersCation Exchangers
 Active ions (counter ions) are cations.
 Consists negatively charged groups and attract positive
charged groups.
 The functional group in cation exchange resins are
usually acids. So it is called as acidic ion exchangers.
 They are usually high molecular weight cross-linked
polymers having sulphonic, carboxylic, phenolic groups
etc with equal amount of cations.
 In cationic exchangers hydrogen ions are mobile and
are exchangeable with other cations and the anions
remain attracted to resin.
 Cation exchange is good for removing metal ions from
an aqueous solution.

• When a salt solution is passed through cation
exchanger, the H+
ions enter the solution and the cations
of the salt get attached to the resin.
Resin-H+
+ M+
→ Resin-M+
+ H+

• Strong cation exchange resins consists of SO3H-
group
and weak cation exchange resins consists of groups like
COOH-
, OH-
, SH-.

e.g. a cation exchanger in the free carboxylic acid form:
X-COO-
H+
X = Frame work (matrix)
-COO-
= Fixed charge (anionic),
Non-exchangeable
H+
= Counter ion (cation), Exchangeable
They are usually (but not always) supplied in the Na+
form:
X-COO-
Na+
or Na +
, Where = Matrix
2 Na+
+ Ca++
Cl2 Ca++
+2 Na+
Cl-
e.g. exchange with CaCl2 aqueous solution

Cation exchangeCation exchange

Cation Exchange ResinsCation Exchange Resins
Trade name Functional group Framework material
Amberlite IR-120 --SO
3
H Styrene/divinylbenzene
Dowex --SO
3
Zerolit --SO
3
SE cellulose --C
2
H
4
--SO
3
H Cellulose
Amberlite IRC-50 --COOH Methacrylic acid
CM --CH
2
COOH Cellulose, fibrous

Anion Exchange ResinsAnion Exchange Resins
• Active ions (counter ions) are anions.
• The functional groups added to the resin is similar to
cation resins but are basic instead of acidic.
• Consists positively charged groups and attract
negatively charged groups. Also called basic ion
exchangers.
• They are usually resins consisting amine or
quaternary ammonium groups as integral part and
equal amount of Ce-
, SO4
2-
, OH-
ions, which are
mobile and exchangeable.

• Quaternary ammonium a strong base --
CH2N(CH3)3+OH-
o CH2N(CH3)3+OH-
+ B-  Res-CH2N(CH3)3
+
Cl-
+ OH-
• Polyalky amine a weak base -- NH(-R)2+OH-
NH(-R)2+OH-
+ B-
 Res-NH(-R)2
+
B- +OH-

The polar groups attached to the matrix are tertiary or
quaternary ammonium groups (basic).
e.g. Anion exchanger in the quaternary ammonium form:
X. NR3+
OH –
X = Framework (matrix)
-NR3 +
= Fixed charge (cationic)
Non exchangeable
-OH–
= counter ion (anion), Exchangeable
or Cl
-
(where, is the matrix)
e.g. exchange with Na2SO4 solution
2 Cl
-
+ Na2
+2
SO4
-2
SO4
-2
+ 2 Na+
Cl -
They are supplied as the chloride rather than the hydroxide as
the chloride form is a more stable. Represented as: X. NR3+
Cl -

Trade name Functional group Framework material
Amberlite IRA-400
-CH2-N+
(CH3)3
Styrene/divinylbenzene
Zerolit FF-IP
-CH2-N+
(CH3)3
Styrene/divinylbenzene

Other types of Ion ExchangersOther types of Ion Exchangers
1. Synthetic inorganic ion exchangers
2. Natural organic ion exchangers
3. Synthetic organic ion exchangers
4. Condensation Polymers
5. Addition Polymers
6. Inorganic exchangers
7. Salt exchangers
8. Bonded exchangers
9. Resinous exchangers

Other types of Ion ExchangersOther types of Ion Exchangers
1. Synthetic inorganic ion exchangers
 Molecular sieves having regular structure and pore
size act as ion exchangers.
 Synthetic exchangers can be prepared from
zirconium and thorium oxides.
2. Natural organic ion exchangers
 Substances like cotton, paper, nut shell can be
converted into cation exchangers by sulphonation
or phosphorylation process.

3. Synthetic organic ion exchangers
 Groups like sulphonic acid and phosphoric acid,
when attached to the polymers give certain
exchange properties.
4. Condensation polymers
 These are high molecular weight cross linked
structures.
 These are formed by splitting out such small
molecules as water, alcohol and ammonia from
small polyfunctional monomers.
 Thus, phenol may condense with formaldehyde to
give a cross linked polymer structure.
 The Condensation Polymers are easily broken up by
hydrolysis and oxidation.

5. Addition polymers
 Addition Polymers are formed from the free radical
by polymerization of mixtures of olefinic and
diolefinic compounds.
 These are resistant to hydrolytic cleavage and
stable to heat and pH changes.
 Thus, styrene may be copolymerised with a small
proportion of divinylbenzene followed by
sulphonation to give a cross linked polymer network.

6. Inorganic exchanger
 Combinations of hydrous oxide with one oxide more
acidic than the other have now been found to
have ion exchanging properties.
 Many combinations of acidic and basic oxides have
been prepared. For examples:
 Titanium arsenide - used to absorb alkaloids
 Hydrous antimony peroxide – used to study
exchange equilibrium of potassium ions with
hydrogen and other ions.

7. Bonded exchanger
 These exchangers are useful in recovering traces of
oxy anions and heavy metals from natural water.
 Some groups that have been bonded are 8-hydroxy
quinoline, weakly basic amino groups, chelating
diamines, dithiocarbamate groups.
8. Salt exchanger
 These exchangers are used for chromatography of
anions as well as cations.
 Resins with non-cyclic, uncharged ions carrying
groups have also been prepared.

9. Resinous exchanger
 A large number of resinous exchangers are
available:
 Polyvinyl alcohol has been cross linked and
provided with various functional groups such as
sulphonic, phosphoric, carboxylic, secondary and
quarternary amines to give large number of new
exchange resins.

Types of ion exchangersTypes of ion exchangers

InstrumentationInstrumentation
• Instrumentation of ion exchange
chromatography includes the following:
 Apparatus
 Types of Resins
 Preparation of Ion Exchange Columns
 Introduction of Sample
 Mobile Phase/Solvents Used
 Elution Techniques
 Detector
 Analysis of the Elute

ApparatusApparatus
• The apparatus is a simple glass tube tapered at the
bottom and fitted with a top.
ION EXCHANGE RESIN
GLASS WOOL
10 CMS TO 100CMS

ApparatusApparatus
• Generally ratio of 10:1 to 100:1 between height and
diameter is used.
• To increase efficiency of separation height can be
increased .
• Based on material to be separated diameter of the
column is selected.
• Columns should neither be too wide or too narrow
• The adsorbent is supported on a plug of cotton or
glass wool.

Types of resinsTypes of resins
• There four basic types of resins commonly used
in ion exchange chromatography.
• Selection of the resin is based on the type of
substance to be separated.
• Resin should have small particle size
Different types of resins are as follows:
1) Strongly Acidic Cation Exchange Resin
2) Weakly Acidic Cation Exchange Resin
3) Strongly Basic Anion Exchange Resin
4) Weakly Basic Anion Exchange Resin

Strongly Acidic Cation Exchange ResinStrongly Acidic Cation Exchange Resin
• Useful between pH range between 1 – 14.
• Used in fractionation of
 Cations
 Inorganic separations
 Lanthanides
 Vitamins
 Peptides
 Aminoacids
Eg. Sulphonated polystyrene resin

Weakly Acidic Cation Exchange ResinWeakly Acidic Cation Exchange Resin
 Cations
 Biochemical separations
 Transition elements
 Antibiotics
 Organic bases
 Aminoacids
Eg. Carboxylic polymethacrylate

Strongly Basic Anion Exchange ResinStrongly Basic Anion Exchange Resin
 Anions
 Halogens
 Fatty acids
 Vitamin B Complex
Eg. Quarternary Ammonium Compounds

Weakly Basic Anion Exchange ResinWeakly Basic Anion Exchange Resin
 Anionic complexes of metals
 Anions of different valencies
 Vitamins
 Aminoacids
Eg. Phenol formaldehyde and polyamine polystyrene
resins

Preparation of the columnPreparation of the column
• Preparation of the column can be done as Wet
Packing
Glass wool or cotton plug is placed in the column
as a support
Slurry of resin is made in water
Remove fine particles by decantation
Column is held in vertical position and slurry of
resin is then poured into column and backwash
with water.

Precautions to be takenPrecautions to be taken
 There should be no air bubbles and swelling effects
must be decreased.
 Slurry should be added in several parts and resin is
allowed to settle between each addition.
 Eluent should be allowed to pass through the
column for certain time.
 Back washing should be done by running up from
the bottom.
 Level of water should never fall below surface of
resin.

Introduction of the sampleIntroduction of the sample
• Solution of the sample which is to be introduced
is prepared.
• Poured on top of ion exchange resin using a
micro syringe or micropipette.
Then the following steps are observed:
Diffusion of ions from sample solution to the
surface of ion exchanger.
Diffusion of ion through matrix structure of ion
exchanger to the exchange site.
Exchange of ions at exchange site.

 Diffusion of exchanged ion to the surface.
 Selective desorption by the eluant and diffusion of
molecule to external solution.
Eg. CATION EXCHANGE RESIN:
+
RESIN
SAMPLE
SOLUTION
DIFFUSION
THROUGH MATRIX
STRUCTURE
ION
EXCHANGE

Mobile phaseMobile phase
• Ions adsorbed by the ion exchanger can be
eluted out using a suitable solvent.
• Sample percolated through column.
• Wash solvent with water or another eluant.
Eluting agents:
1. Cation Exchangers strong acids
RM+H+
RH+ M+
2. Anion Exchangers Alkali
RA+OH- ROH+A-
• Elute can be collected either as a whole or in
fractions

ION EXCHANGE TECHNQUEION EXCHANGE TECHNQUE
• Components can be separated generally by two types
of techniques. They are:
1. Batch Method
2. Column Method

• This is a single step method in which the resin and the
solution are taken into a single container and is mixed
till equilibrium is attained.
• Then, the solution is filtered.
• This method is usually used for softening of water.
• The hardness of water is removed by exchange of Ca2+
and mg2+
ions present in water with Na+
ions of resin.
2RSO3
-
+ Ca2+
(RSO3
-
) Ca2+
+2Na+
• This resin is treated with 15% solution of Nacl to regain
exchange capacity.

• Deionsed or demineralised water can also be
prepared by this method.
1. Cations of electrolytes are replaced by H+
ions
RSO3
-
+ H+
+ M+
RSO3
-
M+
+ H+

2. Water is then treated with anion exchangers

R+
OH-
+ Cl-
R+
Cl-
+ OH-

Finally, H+
+ OH-
H2O

 In this method, the resin is made in to slurry by using
distilled water and is placed in column
chromatography.
 The ratio of the height to diameter of the column is
usually 10:1 or 20:1.
 The column is packed with fine ion exchange resin.
 The particle size 50-100 mesh or 100-200 mesh is used
for analytical separations.
 The resin should be shaken with water in an open
beaker for several minutes and then filled in the
column.
 Since the resin swell in contact with water so it is
advisable not to fill the column with dry resin and then
run water into it; it might break the column
 Care should be taken to prevent the entrapment of air

 The fine particles are removed from the column by
back washing in which water is runned in to the
column both from top and bottom also.
 This back washing is also useful in removal of air
bubbles.
 The particle size of resin used should be small to
provide large surface area for efficient exchange.
 The level of water should be maintained above the
level of resin, to prevent drying of resin.
 The components are separated in the column
according to their selectivity from the resin by
technique like
 Frontal analysis
 Elution analysis
 Displacement analysis

MechanismMechanism
5 distinctive steps:
1. The ion diffuses to the exchanger surface. This occurs
very quickly in homogeneous solutions.
2. The ion diffuses from the matrix surface to the exchange
site, which is based on the degree of cross linkage of
resin and concentration of solution. This step is
considered to be the rate determining step of whole ion
exchange process
3. The exchange of ions takes place at the exchange site,
which occurs instantaneously till equilibrium is attained.
4. The exchanged ion diffuses through the exchanger to
the surface.
5. The molecules are separated by altering the pH or ionic
concentrations or by affinity elution in which the greater
affinity for the exchanger where the bond molecule is

DetectorsDetectors
• The detector which is widely used is electrical
conductivity detector.

Analysis of the eluteAnalysis of the elute
Analysis of the elute is usually done by the following
techniques:
1. Spectrophotometry
Spectrophotometers and colorimeters are used.
2. Polarography
Diffusion of current under constant potential is
measured as a function of time.
3. Conductometry
Electric conductivity of elute from column is
recorded.
4. Radiochemical methods
Radioactivity of elute is automatically recorded by
a. Greiger Muller Counter

Factors affecting the rate of exchangeFactors affecting the rate of exchange
The separation of ions may be influenced by:
1. The nature of ion exchange resin
2. Length of the column
3. Particle size and pH
4. Rate of flow of eluent
5. Temperature

EXCHANGE RESINEXCHANGE RESIN
 After separation of components, the resin is not useful
for another separation.
 It loses its exchangeable functional groups.
 Replacing the exchangeable functional groups does
the regeneration
 Using strong alkalis like NaOH and KOH usually does
regeneration of anion-exchange resins.
 For cation exchange resins strong acids like HCl can be
used.

Factors affecting Ion exchange equilibriaFactors affecting Ion exchange equilibria
1. Nature of the exchanging ions
2. Nature of ion exchange resin
3. Ion exchange capacity

 Nature of the ion is an important factor in the ion
exchange phenomenon.
 Charge of the ion, the radius and the concentration
and affinity of different ions influence the equilibrium
distribution coefficient.
A)At low aqueous concentration and at ordinary
temperatures the extent of exchange increases with
the increasing charge of the exchanging ions
Na+
< Ca2+
< Al3+
< Th4+
B) Under similar conditions and constant charge extent of
exchange depends on the size of hydrated cation.
Thus, for monovalent ions the relative affinity increases
in the following order
Li+
< H+
< Na+
< K+
< Rb+
< Cs+
< Ag+
< Tl+
Lithium is held least strongly on the resin while Thallium is
held most strongly.

For doubly charged ions the ionic size and incomplete
dissociation of their salts are important factors in
determining their relative affinity.
Cd2+
< Be2+
< Mn2+
< Mg2+
C) With strongly basic anion exchange resins, the extent
of exchange for singly charged anions varies with the
size of the hydrated ion in the same way as univalent
cation resins and the relative affinity is
F-
< OH-
< HCO3
-
< Cl-
< HSO3
-
< CN-
< Br-
< NO3
-
< I-
In dilute solutions multicharged anions are generally
adsorbed preferentially.

D) When a cation in solution is being exchanged for
anion of different charge, the relative affinity of ion of
higher charge increases in direct proportion to the
dilution.
Thus to exchange an ion of higher charge on the
exchanger for one of lower charge in solution,
exchange will be favored by increasing the
concentration.
If the ion of lower charge is in the exchanger and the ion
of higher charge is in solution, exchange will be
favored by high dilutions.

 The relative adsorption of ions usually depends on the
presence of functional groups and the degree of cross
linking in the resin.
 The nature of the functional groups largely determines
the type of the exchange phenomenon and their
applications.
 The increase in the degree of cross-linking makes the
resin more selective towards ions of different sizes.
 Ion with the smaller hydrated volume will be adsorbed
preferentially.

 The efficiency of an ion exchange resin is measured by
means of its ion exchange capacity.
 The total ion exchange capacity of a resin may be
defined as the total number of ion active groups per unit
weight of material.
 The greater the number of ions, the greater will be the
capacity.
 It is expressed in terms of milli equivalents per gram of
ion exchange resin.
 The exchange capacity of cation exchange resin is
calculated by determining the number of millieqivalents
of sodium ions adsorbed by one gram of resin in
hydrogen form.
 It is done by passing of sodium chloride of known
concentration in to column containing a known weight
of resin and the eluted from the column is collected and

 If V ml of an alkali solution of strength N in
millieqivalents per liter is required to titrate the acid
eluted from resin weighing W gms.
Ion exchange capacity = v(N)/W
 Similarly, the exchange capacity of an anion
exchanger is determined by passing sodium nitrate
solution of known concentration through column and
the hydroxide ions eluted from the column is titrated
with standard solution of silver nitrate or acid
respectively.

1- Water softening:
o Hardness of water is due to the presence of Ca2+
and
Mg2+
o These ions are removed by passing the water through
cation exchanger charged with Na+
2-Water demineralization:
o Water is first passed through a acidic cationic
exchanger where the metallic cations (Na+
, Ca2+
&
Mg2+
) are exchanged by H+
ions.
o Now it is passed through anionic changer where
anions (Cl-
, No2
-
& So4
2-
) are exchanged by OH-
o H+
& OH-
combine to form water.
3- Neutralization:
Cationic exchanger in [H+] can be used to neutralize alkali
hydroxide & anionic exchanger in [OH+] form to

Applications of Ion Exchange
4. Separation of similar ions from one another:
o Separation is obtained because different ions
undergo exchange reaction to different extent.
o For eg: mixtures of Li+
, Na+
& K+
can be separated by
passing the solution through a cationic exchanger
(0.1N Hcl).
o Mixtures of Cl-
, B-
& I-
can be separated by passing the
solution through a anionic exchanger (0.5N sodium
nitrate).
5. Separation of alkaloids and other nitrogenous
substances from complex samples such as crude
extracts.
6. Separation of metals from mixture.
7. Separation of organic and inorganic ions.

Applications of Ion Exchange ChromatographyApplications of Ion Exchange Chromatography
8. Separation of amino acids.
9. Preparation of pure reagents.
10. Determination of concentrations of traces of ions.
11. Separation of electrolytes from non-electrolytes.
12. Separation of carbohydrates & their derivatives:
 Uronic acids separated on anion exchanger.
 Sugars converted into ionized form by using
borate & separated on strong anion exchanger.
 Hexosamines separated on strong cation
exchanger.

Ion exchange chromatography

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