Gives basic idea about the concept of Ion exchange chromatography.

2. DEFINITION
PRINCIPLE
ION EXCHANGERS
BASIS FOR SEPARATION
INSTRUMENTATION
APPLICATION
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3. ION EXCHANGE CHROMATOGRAPHY:
It is the
based on their affinity towards the ion
exchangers.
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4. Separation is based on the attraction
between oppositely charged particles.
Net charge exhibited by compound is
dependent on their pKa and pH of the
solution in accordance with HENDERSON
HASSELBACH EQUATION.
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5. ION EXCHANGERS
Special type of polyelectrolyte and consist of
to which are bonded a large
number of electrically charged group.
Classification:
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Ionexchanger
Based on the
ionogenic group
Based on the nature
of the source
Based on the
structure
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6. CATIONIC
EXCHANGE RESIN
STRONG WEAK
ANIONIC
EXCHANGE RESIN
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STRONG WEAK
R-SO3H R-COOH R-NH4
+R2-NH
Strong ion exchange media : no variation in charge, sample loading capacity is
maintained
Amberlite IR-120
Dowex
QAE-
Sephadex A-25
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Eg.
Clay,Peat,Lignite,Dolomite,
etc
Eg. sephadex
Eg. Acrylic amide,
formaldehyde, silicates
NATURAL
SEMI SYNTHETIC
SYNTHETIC

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PELLICULAR TYPE MACRORETICULAR
RESIN BEADS
MACROPOROUS
30-40µ
Low exchange
capacityVery Low exchange
capacity
5-10µ
Porous highly
efficient
SULPHONATED
RESINS
Low exchange
capacity

9. PROPERTIES
It must be sufficiently cross linked to have only a
negligible solubility.In order to permit diffusion of ions
through the structure at constant and finite rates
Swollen resin must be denser than water
Resin must be chemically stable
Cross linking is of greater importance
Swelling
polar solvents--- swelling
Non-polar solvents----- contraction
Particle size---- decrease higher the rate of ion exchange
50-100mesh/100-200mesh
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10. BASIS FOR SEPARATION
Capacity factor- The number of sites availbale for
the exchange.
capacity = V X N
W
Flow rate of sample - 1ml/min or 30 drops/min
Particle size- 100-200mesh for resins
pH- if exchange capacity of cationic exchanger
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For cation-5 moles/g
For anion-3.5 moles/g

11. Contd…
Distribution coefficient (KD)= Amount of ions in resin
Amount of ions in solution
Separation factor (α) = KD of component 1
KD of component 2
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12. INSTRUMENTATION
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13. Contd…
•
Pre-packed ion exchange column
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14. PROCEDURE
Buffer selection and preparation
Column and media preparation
Sample preparation
Sample loading
Elution
Re-equilibration
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15. Eg.
In cation exchange chromatography, using a functional group on the solid
support with a pKa of 1.2, a sample molecule with a pI of 8.2 may be run in a
mobile phase buffer of pH 6.0.
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As a rule, the pH of the mobile phase buffer must be between the
pI (isoelectric point) or pKa (acid dissociation constant) of the
charged molecule and the pKa of the charged group on the solid
support.
USUALLY PREPARED VOLUME – 500ml

16. BUFFER SYSTEM FOR ANIONIC EXCHANGE
RESINS
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17. BUFFER SYSTEM FOR CATIONIC EXCHANGE
RESINS
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18. Equilibrate column with 5–10 column
volumes of start buffer or until the baseline,
eluent pH and conductivity are stable.
Using prepacked columns is highly
recommended to ensure the best
performance and reproducible results. An
evenly packed column ensures that
component peaks are not unnecessarily
broadened as sample passes down the
column so that the best resolution can be
achieved.
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19. Contd…
Allow buffers, media or prepacked columns to reach the same
temperature before use.
Rapid changes in temperature, for example removing packed
columns from a cold room and then applying buffer at room
temperature, can cause air bubbles in the packing and affect
the separation.
Wash away storage solutions and preservatives before using
any IEX medium.
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20. Contd..
Increase the volumes used for column equilibration before
the first run if using buffers containing detergents or a
different counter-ion to the one in which the medium has
been stored.
The volume required for the packed bed is determined by the
amount of sample to be purified and the binding capacity of
the medium.
Pack a column that will have approximately 5-fold excess of
the binding capacity required with a bed height up to 20 cm.
Wet packing
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21. Desalt the sample and transfer to the buffer
Adjust the sample to the chosen starting pH and ionic
strength and apply to the column.
Sample volume should be based on the capacity of ion
exchange resins
For protein samples maximum concentration of about 50-
70mg/ml can be used
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22. Apply samples directly to the column via a chromatography
system, a peristaltic pump or a syringe.
The choice of equipment depends largely on the sample
volume, the size of column, the type of IEX medium and the
requirements for accuracy in gradient elution.
Ensure that the top of the column bed is not disturbed during
sample application
Do not change buffer conditions until all unbound material
has been washed through the column (monitored by UV
absorbance) and until UV and conductivity values have
returned
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23. Achieved by decreasing the affinity of solute. It is done by
using salt of varying concentration
Bound proteins are eluted by controlled changes in ionic
strength or pH. The way in which these changes take place, by
using a linear or step elution, is selected according to the aim
of the separation:
Linear gradient elution
–high resolution separation or analysis
Step elution
–faster separation time, reduced buffer consumption
–group separation
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24. Linear gradient elution:
Begin elution using a linear gradient volume of 10–20
column volumes with an increasing ionic strength up to
0.5 M NaCl (50%B).
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25. Step elution
Elute bound proteins with 5 column volumes of
start buffer + NaCl at chosen ionic strength.
Repeat at higher ionic strengths until the target
protein(s) has been eluted.
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26. Wash with 5 column volumes of 1 M NaCl (100%B) to elute any remaining
ionically-bound material. Include a wash step at the end of every run in order
to remove any molecules that are still bound to the medium. Monitor UV
absorbance so that the wash step can be shortened or prolonged, as
necessary
Re-equilibrate with 5–10 column volumes of start buffer or until eluent pH
and conductivity reach the required values. A re-equilibration step after
washing returns the column to start conditions before applying further
samples. Whenever possible, monitor pH and conductivity to check when
start conditions have been reached. The re-equilibration step can then be
shortened or prolonged as necessary
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27. SPECTROPHOTOMETRIC METHOD
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FLAME PHOTOMETRY
CONDUCTOMETRIC METHODS
RADIO ISOTOPE METHOD

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29. APPLICATION
• Separation of similar ions from one another
• Removal of interfering radicals
• Softening of hard water
• Complete demineralisation of water
• Separation lanthanides and actinides
• Separation of sugars
• Separation of amino acids
• Preparation of pure reagents
• Hydrometallurgy
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31. REFERENCE
www.harvardapparaturs.com, guide to ion
exchange chromatography
Instrumental analysis of chemical analysis by
Gurudeep R.Chatwal, Sham.K.Anand, Pg. 2.662-
2.672
Instrumental methods of chemical analysis by
B.K.sharma, C-123 -150
http://www.gelifesciences.com/file_source/GELS
/Service%20and%20Support/Documents%20and
%20Downloads/Handbooks/pdfs/Ion%20Exchang
e%20Chromatography.pdf
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