Download and play it my friends it contain VIDEO The technique of ion exchange chromatography is based upon the interaction between charged solute molecules and oppositely charged moieties covalently linked to chromatographic matrix. The reasons for its widespread success is its applicability, high resolving power, high capacity and simplicity of the technique. Separation in ion exchange chromatography depends upon the reversible adsorption of charged solute molecules to immobilized ion exchange groups of opposite charge. Most experiments are performed by following : Video For Understanding Play It

1. ION - EXCHANGE
CHROMATOGRAPHY
Shrikant R. Divekar
M.Pharm
Pharmaceutics
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2. INTRODUCTION
• The technique of ion exchange chromatography is
based upon the interaction between charged
solute molecules and oppositely charged moieties
covalently linked to chromatographic matrix.
• The reasons for its widespread success is its
applicability, high resolving power, high capacity
and simplicity of the technique.
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3. • SEPARATION IN ION EXCHANGE CHROMATOGRAPHY DEPENDS UPON THE
REVERSIBLE ADSORPTION OF CHARGED SOLUTE MOLECULES TO IMMOBILIZED
ION EXCHANGE GROUPS OF OPPOSITE CHARGE. MOST EXPERIMENTS ARE
PERFORMED BY FOLLOWING : VIDEO FOR UNDERSTANDING PLAY IT
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6. • Separation is obtained since different substance
have different degrees of interaction with the ion
exchanger due to differences in their charges,
charge density, partical size and distribution of
charge on surface. These interactions can be
controlled by varying conditions of pH and ionic
strength.
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7. THE MATRIX AND IT’S AFFINITY
• An ion exchanger consists of a water insoluble
solid of complex structure capable of exchanging
counter ions with ions in the surrounding medium
(Analyte in mobile phase) in a reversible process.
• The charged groups of matrix are associated with
mobile counter ions. These can be reversibly
exchanged with other ions of same charge
without altering the matrix. 7

8. • It is possible to have both positively and negatively
charged exchangers as shown above.
• Positively charged exchangers have negatively charged
counter-ions(anions) available for exchange and are
called anion exchangers.
• Negatively charged exchangers have positively charged
counter-ions(cations) and are termed as cation
exchangers.
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9. • Presence of charged groups is fundamental of an ion
exchanger. The type of group determines the type and
strength of the exchanger while their total number and
availability determines the ion exchange capacity.
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10. ION EXCHANGE CAPACITY
• Total ion exchange capacity of resin is dependent
upon the total number of ion active groups per unit
weight of resin. Greater the number, greater is the
capacity of resin.
• This capacity is usually expressed as milliequivalent
per gram of exchanger.
• The capacities of weak acidic cation exchange or
resin also depend upon pH. Good values are given at
about pH 9.0 for weakly acidic or pH 5.0 or below for
weakly basic resins. 10

11. • The capacity of an ion exchanger is a quantitative
measure of its ability to take up exchangeable counter
ions and is therefore important. The capacity may be
expressed as total ionic capacity, available capacity or
dynamic capacity.
• The total ionic capacity is the number of charged
substituent groups per gram dry exchanger or per ml of
swollen gel. The total capacity can be measured by
titration with a strong acid or strong base.
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12. • The available capacity for the gel is the actual amount of
analyte that can be bound to an ion exchanger, under defined
experimental conditions.
• If the defined conditions include the flow rate at which the
gel was operated then the amount bound is referred to as
dynamic capacity for the ion exchanger.
• Available and dynamic capacities depend upon
a) Properties of analyte.
b) Properties of ion exchanger.
c) The chosen experimental conditions.
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13. PROPERTIES OF ANALYTE
• The properties of analyte which determines the available or
dynamic capacity on a particular ion exchange matrix are its
molecular size and its charge/pH relationship.
On a porous matrix,molecules which are small enough to
enter the pores will exhibit higher available capacity than
those molecules which are restricted to charged
substituents on surface of gel.
Similarly, since the interaction is ionic, the analyte’s
charge/pH relationship must be such that the protein carries
the correct net charge, at sufficiently high surface charge
density, to be bound to a particular ion exchanger under the
chosen buffer conditions.
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14. PROPERTIES OF ION
EXCHANGER
• The properties of ion exchange matrix which determine its
available capacity for a particular protein are the exclusion limit
of matrix and type and number of charged substituent.
High available capacity is obtained by having a matrix which is
macroporous and highly substituted with ionic groups which
maintain their charge over a wide range of experimental
conditions.
Non-porous matrices have considerable lower capacity than
porous , but higher efficiency due to shorter diffusion
distances. 14

15. EXPERIMENTAL CONDITIONS
• The experimental conditions which affect the observed
capacity are pH, ionic strength of buffer, nature of
counter-ion, flow rate (esp. for dynamic capacity which
decreases as flow rate is increased) and temperature.
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16. PHYSICAL PROPERTIES OF ION
EXCHANGE RESINS
• Cross Linking:
This affects swelling and strength of ion exchange resin by its
degree of cross linking. As cross linking decreases, swelling
increases.
Solubility is also greatly affected. If polystyrene is cross linked by
incorporation of divinyl benzene, the mechanical strength is
imparted to the resin and makes it insoluble in common solvents.
• Particle size and porosity:
Surface area contributes directly to the rate of exchange. Large
surface area and small particle size will increase the rate of ion
exchange. Ion exchange resins are stable towards strong acids,
strong bases and organic solvents. Particle range 50-100 mesh or
100-200 mesh is most commonly employed. 16

17. • Swelling:
When resin swells, the polymer chain spreads apart which forms narrow
passage throughout the resin bed.
In polar solvents swelling occurs while in non-polar solvents contraction occurs
• Regeneration:
Ion exchange resins after use get deactivated as the replacement of ion takes
place.
In cation exchange resin, cations from the given solution get attached to the
resin and deactivation occurs.
So the cation exchange resins are regenerated by treatment with aqueous acid
followed by washing with water.
The resin gets converted to H+
form and can be used for analytical purpose.
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18. MECHANISM OF ION EXCHANGE
PROCESS
• The ion exchangers behave as a porous network
which carry a surplus electric charge distributed over
the surface and throughout the pores. The surplus
charge is compensated by ions of opposite charge.
• When the ionization takes place they are exchanged
with the ions of opposite charge which migrate into
the solution.
• In this process, chemical bonds are not formed but
exchange occurs by diffusion in two different stages.
a)Film diffusion.
b)Particle diffusion.
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19. a) Film Diffusion:
It is a diffusion of counter ions through a surface liquid film
which surrounds the ion exchanger. The film is extremely
thin and is prominent in dilute solutions and has smaller
counter ions.
b)Particle Diffusion:
It refers to diffusion of counter ions within the pores of
exchanger. It is predominant at high concentration and
with large ions. This is increased by exchangers with low
degree of cross linking, high exchange capacity, Counter
ions with low valency and increasing temperature.
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20. APPLICATIONS
• Separation of similar ions
• Softening of hard water
• Complete demineralization of water
• Purification of organic compounds
• Separation of sugars
• Separation of amino acids
• Purification and recovery of pharmaceuticals
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21. ION PAIR CHROMATOGRAPHY
• Developed by Dr. Gordon Schill and coworkers in 1973 and
is an alternative method to ion exchange chromatography.
• It is a HPLC technique used for separation of charged
analytes in which formation of ion pair takes place with
addition of counter ion and then this neutral ion pair
complex undergoes partitioning between stationary and
mobile phase.
• The technique can be named in several ways-Solvophobic
ion chromatography, Counter ion chromatography,
Surfactant chromatography and Ion association
chromatography.
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22. • Ion pair chromatography can be performed with
 Normal phase columns.
 Reverse phase columns.
Normal phase:
Ion pair Reagent + aq. Buffered solution coated on silica gel to
form S.P.
Mobile phase is organic solvent.
However, it involves sample transfer to organic phase prior to
analysis and most of the polar compounds are less soluble in it.
Reverse phase :
Employs permanently bonded alkyl layer and hence preferred
over normal phase columns.
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23. ION PAIR REAGENT
• It is a large organic counter ion, which is added
to mobile phase at low concentration.
• It itself is charged and used to separate organic
solute ions of opposite charge by forming ion
pair complex.
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24. • Following are some ion pair reagents used
For basic compounds: Cationic samples separated by
addition of hydrochloric, perchloric, and perfluorocarboxylic
acids and straight chain alkyl sulphonic acids to the mobile
phase and a pH of 3-4.
For HPLC-MS technique we use volatile trifluoroacetic acid,
heptanefluorobutyric acid.
For acidic compounds: Anionic samples separated with
straight chain alkyl quaternary ammonium salts or alkyl
amines. pH of the mobile phase is about 7. 5 24

25. ADVANTAGES OF ION PAIRING
• Suitable for simultaneous analysis of non-ionic and ionized species.
• Ion pair formation is relatively faster (within seconds) and is carried
out at room temperature.
• It is suitable for compounds like tertiary amines, which are difficult
to derivatize into suitable products for detection purposes.
• This allows sensitivity to be manipulated through changes in
composition of the mobile phase alone.
• Gives sharp peak shapes and highly reproducible results.
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26. THE END
Thank You !!!
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