Ion exchange chromatography is a technique used to separate charged molecules based on their interaction with oppositely charged groups on a resin. It works by reversible exchange of ions between the ions in a sample and those on an ion exchange resin. There are two types of resins - cation exchange resins which interact with positively charged ions, and anion exchange resins which interact with negatively charged ions. The process involves equilibrating the resin, applying the sample, washing unbound molecules, and then eluting the bound molecules using an increasing salt gradient. Ion exchange chromatography is widely used to purify proteins and analyze ions in applications like biochemistry, water quality testing, and metal purification.

1. Presented by,
R . Harishma .

2.  Analytical technique.
 Used to separate the mixture of chemical
substance.
 All forms of chromatography works on a same
principle.
 They all have a
Stationary phase (solid or liquid)
Mobile phase (liquid or gas)

3.  It is a type of column chromatography.
 In chromatography is the process of separation
of molecules from a mixture.
 These molecules are in different types,
macro molecules( protein, nucleic acid)
smaller molecule(ions)
 In these chromatography we separate charged
molecules (or) polar molecules.

5.  In which molecules are separate based on their
charge.
 Stationary phase surface displays ionic
functional groups that interact with analyte
ions of opposite charge.
 Also known as cation – anion exchange
chromatography.

6.  It is useful and popular method due to its,
High capacity
High resolving power
Mild separation conditions
 Some general terms are,
Eluent – fluid that enter into the column
Eluate – fluid that leaves the column
Analyte/ sample

7.  Exchanging of charged molecules for the
mobile phase
 Separation occurs by reversible exchange of
ions between the ions present in the solution
and those present in the ion exchange resin.
 Loading buffer has low conductivity in order to
promote interactions, between the sample and
the resin.

8.  There are two types of ion exchange resin used,
Cation exchange resin
Anion exchange resin
 Strong cation exchange resins typically have –
sulfonic acid functional group
Weak cation resin – carboxylic acids

9.  Strong anion exchange resin typically have
quaternary amines
weak anion resin – secondary/tertiary
amines.
 The term strong and weak refers to the
acid/base properties of column material.
 Weak resins are charged over a smaller pH
range than strong resins.

10.  It have stationary phase and mobile phase ,
that are always interact with each other.
 The interaction between stationary phase and
mobile phase are varies depends on the
different molecules.
 In these case of ion exchange chromatography
we run a column.
 Stationary phase contains a polymeric
materials.

11.  Mobile phase contains charged molecules.
 In cation exchange chromatography,
In anion exchange chromatography,

12.  To elute the sample of interest salt gradient is
used.
 Samples that are less tightly bound to the resin
will elute first , samples that are more tightly
bound will elute later , when higher amount of
salt are used.
 The pH needs to be maintained.

13.  In cation exchange chromatography ,
Raising the pH will cause the analyte to be
less positively charged and less likely to interact
with negatively charged resin.
 In anion exchange chromatography ,
Lowering the pH will cause the analyte to be
less negatively charged and less likely to interact
with positively charged resin.

14.  Column material & dimensions:
Material :
Glass – high quality stainless steel (or)
polymers.
Dimensions:
Length : diameter ratio of 20:1 to 100:1

15.  Stationary phase:
composed of two structural elements,
Charged groups
Matrix
 Materials:
Cellulose
silica
polyacrylamide
agarose

16.  Mobile phase:
consists of solution.
Some eluent additives are used,
EDTA
Glycerol
Glucose
Lipid
Urea
Organic solvents

17.  Buffers:
pH value is important parameter for
separation which is controlled by means of buffer.
Cation – citric acid, lactic acid , acetic acid ,
formic acid.
Anion – piperazine , N-methyl piperazine ,
ethanolamine , triethanolamime.

18. These are ,
strong cation resin
weak cation resin
strong anion resin
weak anion resin
General requirements of resins:
should be chemically stable
insoluble in most of common solvents

19. must contain sufficient number of ion
exchange groups.
should have high degree of exchange of ions.
Steps in ion exchange separation:
1. equilibration:
The first step is the equilibration of the
stationary phase to the derived start condition.

20. when equilibrium is reached, all stationary phase
charged groups are bound with exchangeable counter
ions Such as chloride or sodium.
Sample application and washing:
The goal in the step is to bind the target
molecule and wash out all unbound materials.
The sample buffer should have the same pH
and ionic strength as the start buffer in order to bind
all charged target protein.
Charged proteins bind to ionic groups of the
IEX medium.
Uncharged proteins pass through the column
at the same speed as the flow of buffer.

21. Elution:
All samples has been loaded and the column washed
with start buffer. So that all non binding protein have
passed through the column.
The bound proteins are eluted by increasing the ionic
strength of the buffer.
Ionic strength increases one or more bound particles are
elutes and move down the column.
The proteins with lowest net charged will be first once
eluted from the column as ionic strength increases.
The proteins with the highest charge are will be eluted
last.
To elute this by controlling charges in ionic strength
using different form of gradient, proteins are eluted
differently in a purified, concentrated form.

22. Regeneration:
A final wash with high ionic strength
regenerator the column and removes any
molecules still bound.
So the full capacity of the stationary
phase is available in for the next run.
The column is the re equilibrated in
start buffer before starting the next run.

24. APPLICATION:
 It is widely used in biochemistry to isolate
of purify protein sample.
 Anion exchange used to measure the
concentration of anions, including sulfates,
nitrates, nitrites, Fluoride and chloride.
 Cation-sodium, potassium, calcium and
magnesium.
 It is also used in water pollution.
 Metal purification.