A chromatographic Principle

1. Kuldeep Sharma
B.Tech Biotechnology
IV SEM
Amity Institute of Biotechnology

2. Contents
• Introduction
• Principle
• Working
• Ion Exchange Resin
• Exchange Medium
• Preparation of Ion Exchangers
• Advantages
• Applications

3. Introduction
• “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
compounds like proteins differing by only one charged amino acid.
• In Ion exchange chromatography technique one can choose
whether to bind the substance of interest and allow the
contamination to pass through the column and vice versa.
• This technique has been developing 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.

4. Types
Laboratory Commercial Automated

5. Principle
• Ion exchange chromatography relies on the attraction between
oppositely charged stationary phase, known as an ion exchanger, and
analyte.
• The ion exchanger consists of an inert support medium coupled
covalently 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.
• Thus if anion exchange chromatography is performed, negatively
charged sample components will interact more with the stationary
phase and will be exchanged for like charged ions already bounded to
the matrix.

6. 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+
• The remaining neutral and negatively charged particles

7. Working

8. Working
• Bounded interest of ion (X+
) can now be eluted by either
of the two ways;
1. By adding a component M+
having magnitude of charge
more than that of X+
so that M+
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.

9. Regeneration
• Cation exchange resins are regenerated
by treatment with acid, then washing with
water.
• Anion exchange resins are regenrated by
treatment with NaOH, then washing with
water.

10. Factors Affecting Chromatographic
Separation
1. Ion Exchange Resin:
The swelling factor and cross linking is important for the effective
separation.
The cross linking should be controlled as its affects the exchanger’s
capacity.
Swelling helps in proper exposure of charged functional groups for
exchange of ions.
2. Sample:
The concentration and charge of ions
3. Buffer:
The pH of the buffer should impart the same charge to the sample ions as
present in the Column.
Anionic Exchange Chromatography should be carried out with cationic
buffers and vice versa because buffer ion will indulge in ion exchange,
which will be of no use.

11. 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

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

13. 1. Polystyrene
• Polystyrene resins are prepared by polymerisation reaction of styrene and
divinylbenzene.
• Higher concentration of divinylbenzene produces higer cross linkages.
• Polystrene resin are very useful for separating small molecular weight
compounds, however, unsatisfactory for the separation of macromolecules

14. 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.

15. Exchange Medium
• The choice of ion exchangers depends upon the stability,
molecular weight, and ionic strength of the sample
components.
• The volume of exchanger used for separation is usually 2.5
fold greater than to exchange with the ion in the sample.
• The ion exchanger are packed in column having suitable
buffer.
• The ion exchangers are of two types;
1. Anion Exchangers
2. Cation Exchangers

16. 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
Mobile counter ion
Positively charged exchangers
Ion exchanger resin

17. Examples: Anion Exchangers
Source: Wilson, K., & Walker, J. (Eds.). (2010). Principles and techniques of
biochemistry and molecular biology. Cambridge University Press.

18. 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
Negatively charged exchangers
Mobile counter ion
Ion exchanger resin

19. Examples: Cation Exchangers
Source: Wilson, K., & Walker, J. (Eds.). (2010). Principles and techniques of
biochemistry and molecular biology. Cambridge University Press.

20. 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 (0.5 N HCl) and then with base (0.5 N NaOH). Reverse is true
for preparation of cation exchangers.
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,.

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

22. 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