ION EXCHANGE

CHROMATOGRAPHY

S.VITHYA
M.PHARM I YEAR

CONTENTS
DEFINITION
PRINCIPLE
ION EXCHANGERS
BASIS FOR SEPARATION
INSTRUMENTATION
APPLICATION

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DEFINITION
ION EXCHANGE
CHROMATOGRAPHY:
It is the reversible process of
separation of ions based on their
affinity towards the ion exchangers.

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PRINCIPLE
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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ION EXCHANGERS
Special type of polyelectrolyte and consist of three
dimensional polymeric hydrocarbon network to
which are bonded a large number of electrically
charged group.

Ion exchanger

Classification:

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Based on the
ionogenic group
Based on the
nature of the
source
Based on the
structure

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BASED ON THE
IONOGENIC
GROUP
CATIONIC
EXCHANGE
RESIN
STRONG

WEAK

ANIONIC
EXCHANGE
RESIN
STRONG

Amberlite IR-120
Dowex

R-SO3H

WEAK

QAESephadex
A-25

R-COOH

R2-NH

R-NH4+

Strong ion exchange media : no variation in charge,

sample loading capacity is maintained
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BASED ON THE SOURCE

NATURAL

Eg.
Clay,Peat,Lignite,Do
lomite, etc

SEMI
SYNTHETIC

Eg.

SYNTHETI
C

Eg. Acrylic amide,

formaldehyde,
silicates

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sephadex

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BASED ON THE STRUCTURE

PELLICULAR TYPEMACRORETICULMACROPOROUS
AR RESIN
BEADS
30-40
5-10
Low
Porous highly
Very Low
exchange
efficient
exchange
capacity
capacity

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SULPHONATE
D RESINS
Low exchange
capacity

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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BASIS FOR SEPARATION

Capacity factor- The number of sites
availbale for the exchange.
For
moles/g
capacity = V
X cation-5
N
For anion-3.5 moles/g
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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Contd
Distribution coefficient (KD)= Amount
of ions in resin
Amount of
ions in solution

Separation factor () = KD of
component 1
KD of
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INSTRUMENTATION

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Contd

Pre-packed ion exchange column

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PROCEDURE
Buffer selection and preparation
Column and media preparation
Sample preparation
Sample loading
Elution
Re-equilibration

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BUFFER SELECTION

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
Eg.
the solid support.
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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USUALLY PREPARED VOLUME 500ml

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BUFFER SYSTEM FOR ANIONIC

EXCHANGE RESINS

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BUFFER SYSTEM FOR CATIONIC

EXCHANGE RESINS

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COLUMN AND MEDIA

PREPARATION

Equilibrate column with 510

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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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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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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SAMPLE PREPARATION
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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SAMPLE LOADING
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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ELUTION
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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Linear gradient elution:

Begin elution using a linear gradient volume
of 1020 column volumes with an increasing
ionic strength up to 0.5 M NaCl (50%B).

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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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RE-EQUILIBRATION
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 510 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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ANALYSIS OF ELUENT
SPECTROPHOTOMETRIC METHOD
FLAME
PHOTOMETRY
CONDUCTOMETRIC
METHODS
RADIO ISOTOPE
METHOD

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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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Purification
of protein
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Demineraliza
tion of water
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REFERENCE
www.harvardapparaturs.com, guide to ion
exchange chromatography
Instrumental analysis of chemical analysis by
Gurudeep R.Chatwal, Sham.K.Anand, Pg. 2.6622.672
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
%20Exchange%20Chromatography.pdf
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