Sakeena Asmi MSc Biotechnology

1. ION EXCHANGE
CHROMATOGRAPHY
SAKEENA ASMI
MAHATMA GANDHI
UNIVERSITY

2. • The first detailed description of chromatography is generally
credited to Michael Tswett, a Russian biochemist, who separated
chlorophyll from a mixture of plant pigments in 1906.
• Because of the nature of the pigments in the sample, each band had
a distinctive color.
• Thus the name of the process was coined from the Greek words for
color (chromo) and to write (graphy).
• If the individual components of a mixture have widely dissimilar
physical and chemical properties, it is very easy to separate one
from another.
CHROMATOGRAPHY

3. • But as the individual components of a mixture get more and more
similar in physical and chemical properties, it becomes increasingly
difficult to separate them from one another.
• However it can be readily achieved using chromatography.
• The feature common to them all is that two mutually immiscible
phases are brought into contact with each other.
• One of these phases is stationary, while the other is mobile.
• The mobile phase either moves over the surface or percolates
through the interstices of the stationary phase.

4. PARTITION
COEFFICIENT• Partition Coefficient (also known as distribution coefficient) is a
definitive term normally used to describe the way in which a given
compound distributes or partitions itself between two immiscible
phases, the stationary and the mobile phase.
K = Cs
Cm
where Cs and Cm are the concentrations of the compound in the
stationary and the mobile phases respectively.
• This concept of Partition Coefficient is the basic principle of all
chromatographic methods.

5. TECHNIQUES OF
CHROMATOGRAPHY
• There are two basic techniques of chromatography : plane
chromatography and column chromatography.
PLANE CHROMATOGRAPHY
• There are two variations of plane chromatography: paper
chromatography and thin layer chromatography.
• In paper chromatography the stationary phase is supported by
cellulose fibres of the paper sheet.
• In thin layer chromatography the stationary phase is coated onto a
glass or plastic surface.

6. COLUMN CHROMATOGRAPHY
• In column chromatography the stationary phase is packed into a
glass or metal column.
• The stationary phase is either coated onto discrete small particles
(the matrix) and packed into the column or applied as a thin film to
the inside wall of the column.
• As the eluent flows through the column the analytes separate on the
basis of their distribution coefficients and emerge individually in
the eluate as it leaves the column.

7. Avinash Upadhyay - Biophysical
Chemistry-Principles & Techniques
LIQUID

8. ION EXCHANGE
CHROMATOGRAPHY

9. INTRODUCTION
• This technique has been developed since 19th century which was
firstly used for purifying the drinking water.
• Ion exchange chromatography is a distinct principle of
chromatography performed in the column.
• Ion exchange chromatography may be defined as the reversible
exchange of ions in the solution with ions electrostatically bound to
some sort of insoluble matrix or a stationary phase.
• This technique is extremely useful in the separation of charge
compound like proteins differing by only one charged amino acid.

10. TYPES
LABORATORY COMMERCIAL AUTOMATED

11. PRINCIPLE
• Ion exchange chromatography relies on the attraction between
oppositely charged stationary phase, known as an ion exchanger and
analyte.
• It is frequently chosen for the separation and purification of
proteins, peptides, nucleic acids, polynucleotides and other charged
molecules, mainly because of its high resolving power and high
capacity.
• There are two types of ion exchanger, namely cation and anion
exchangers.
• Cation exchangers possess negatively charged groups and these will
attract positively charged cations. These exchangers are also called
acidic ion exchangers.

12. PRINCIPLEContinued…
• Anion exchangers have positively charged groups that will attract
negatively charged anions. The term basic ion exchangers is also
used to describe these exchangers.
• The ion exchanger consists of an inert support medium coupled to
positive (anion exchanger) or negative (cation exchanger)
functional groups.
• To these covalently bound functional groups the oppositely charged
ions are bounded (mobile counter ion), which will be exchanged
with like charge ions in the sample having charge magnitude more
than the ions bounded to the matrix.

13. WORKING
• Consider a column having E- Y+ cation exchanger in which E- is
negative charged exchanger and Y+ is the mobile counter ion.
• Let X+ be the cation in the sample having charge greater than Y+ .
• The X+ ion can exchange sites with the counter ion Y+ with
satisfying the following relationship :
E- Y+ + X+ E- X + + Y+

14. Continued…
Ion Exchange Chromatography (Cation Exchange)

15. Continued…
• Bounded interest of ion (X +) can now be eluted by either of the two
ways;
1. By adding the component Y+ having magnitude of charge more
than that of X + so that Y+ will replace X + and X + will be eluting
out.
2. By changing pH of the solvent (mobile phase) so that X + have no
charge and is then unbounded from the matrix and can be eluted
out.

16. Ion Exchange Chromatography (Anion Exchange)

17. ION EXCHANGERS
• An ion exchanger consists of two basic components :
1. Ion exchange resin
2. Exchange medium (Cation exchanger and Anion exchanger)
- Charged functional group.
- Mobile counter ion.

18. ION EXCHANGE RESIN
 Two main groups of materials are used to prepare ion exchange
resins :
• Polystyrene
• Cellulose
 Resin made from both of these materials differ in their flow
properties, ion accessibility and chemical and mechanical stability.
 Selection of one or the other type of resin is done on the basis of
compounds being separated.

19. 1. POLYSTYRENE
• Polystyrene resins are prepared by polymerisation reaction of
styrene and divinyl benzene.
• Higher concentrations of divinyl benzene produces higher cross
linkages.
• Polystyrene resins are very useful for separating small molecular
weight compounds, however, unsatisfactory for the separation of
macromolecules.

20. 2. CELLULOSE
• Cellulose is a high molecular weight compound which can
be readily obtained in a high pure state.
• Cellulose has much greater permeability to
macromolecules.

21. EXCHANGE MEDIUM
• The choice of ion exchangers depends upon the stability, molecular
weight and ionic strength of the sample components.
• The ion exchanger are packed in column having suitable buffer.
• The ion exchangers are of two types :
1. Anion Exchangers
2. Cation Exchangers

22. 1. Anion Exchangers
• The anion exchangers have positively charged exchanger with
negatively charged mobile counter ion available for exchange.
• If the basic functional groups are introduced, the resin becomes
anion exchanger.
• Tertiary amines Strong anion exchangers
• Secondary amines Weak anion exchangers

23. Examples : Anion Exchangers
Source : Wilson K & Walker J (2010). Principles and Techniques of Biochemistry and
Molecular biology.

24. 2. Cation Exchangers
• The cation exchangers have negatively charged exchanger with
positively charged mobile counter ion available for exchange.
• If acidic functional group are introduced, then the resin becomes
cation exchangers.
• Sulphonic acid Strong cation exchangers
• Carboxylic acid Weak cation exchangers

25. Examples : Cation Exchangers
Source : Wilson K & Walker J (2010). Principles and Techniques of Biochemistry and
Molecular biology.

26. PREPARATION OF ION
EXCHANGERS
• Preparation of the exchange medium is essential for satisfactory
performance of ion exchange chromatography.
• Apart from removing impurities, there are three major steps that are
absolutely important in ion exchanger preparation.
1. SWELLING: For anion exchangers it is done by treating it first
with an acid (0.5N HCl) and then with base (0.5N NaOH).
Reverse is true for preparation of cation exchangers.

27. 2. REMOVAL OF FINE PARTICLES: Large number of such
particle can result in decreased flow rate and improper resolution.
For this the exchangers are repeatedly suspended in large volume of
water.
3. ADDITION OF COUNTER IONS: Accomplished by washing
the exchanger with suitable reagent depending upon which counter
ion to be introduced.
For e.g : NaOH (Na+), HCl (H+) etc…

28. CHOICE OF BUFFER
• The choice of buffers which maintain the pH of the column is
dictated by the compounds to be separated and the type of ion
exchange being carried out (anionic or cationic).
• Anion exchange chromatography should be carried out with
cationic buffers. Reverse is true for cation exchange
chromatography.
• If anion exchange or cation exchange is carried out with anionic or
cationic buffers respectively, the buffer ions will indulge in ion
exchange and hamper sample component exchange.
• The pH of the buffer should impart the same charge to the sample
ions as is present on the counterion.

29. Some Volatile Buffers Used in Ion-Exchange
Chromatography
Avinash Upadhyay - Biophysical Chemistry-Principles & Techniques

30. APPLICATIONS
• Softening of hard water.
• Demineralization of water.
• To analyze base composition of nucleic acid.
• To concentrate the metal ions in the sample.
• To measure the additives in food and drug sample.
• To separate protein mixtures.

31. ADVANTAGES
• Detectability: useful for the detection of many inorganic salts and
also for the detection of organic ions with poor UV absorptivity
like alkyl amines or sulfonates.
• Preparative separations: usually preferred because of the
availability of volatile buffers. Volatile buffers makes the removal
of mobile phase easier.
• Useful to resolve very complex samples, i.e in the case of multi
step separation.
• Useful for separation of mixtures of biological origin, in organic
salts and some organo – metallics.

32. • Cost effective
• Re-usable
• Easily collectable
• Low maintenance cost
• Efficient technique
• Quick separation

33. DISADVANTAGES
• Column efficiency is less.
• It is difficult to achieve control over selectivity and resolution.
• Stability and reproducibility of the columns become questionable
after repeated use.

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THANK