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| Column Chromatography: Ion - exchange Chromatography: Types, Principle, Application, Advantages and Disadvantages |
Column
Chromatography: Ion - exchange Chromatography: Types, Principle, Application,
Advantages and Disadvantages
Column
Chromatography: It refers to the separation of different types of
proteins from each other based on two phases –
a) Solid
stationary phase
b) mobile
phase
Ion – exchange Chromatography
It separates molecules based on their charge
because of its high resolving power and high capacity.
Types of
Ion – exchange Chromatography:
Two types
-
a) cation
exchange
b) anion
exchange
Principle
of Ion - exchange Chromatography:
1. It is
based on the reversible electrostatic attraction of a charged molecule to a
solid matrix that contains covalently attached groups of opposite charge.
2. Cation
exchangers posses negative groups and attract positive cations (acidic).
3. Anion
exchangers contain positive groups and attach to negative groups, anions. They are basic groups.
4. The
cation exchangers result from ionization of acidic groups and anion exchangers
result from basic groups.
5. During
the cation-exchange process, positively charged ion-exchange matrix by
displacing the counter ion, which is bound to the resin by electrostatic
attraction.
6. Elution
is achieved using a salt containing buffer. The salt cation displaces the
protein from ion-exchange matrix.
7. In the
case of negatively charged proteins, an
anion exchanger is employed with the protein adsorbing to the column by
replacing a negatively charged counter ion.
8. Proteins
may be subsequently eluted by altering the pH or by increasing the salt
concentration of buffer.
Cation
exchangers:
Sulfate derivatives, carboxy-methyl groups.
Anion exchangers:
Amino ethyl and di-ethyl-amino ethane.
Salt-containing buffer:
Imidazole, Bis-tris buffer.
Negatively
charged proteins:
Aspartic, glutamic acids.
Positively charged proteins:
Arginine, histidine, lysine.
Charge of
proteins depends on -
a) pH of
protein solution
b)
N-terminal and C-terminal carboxy groups
c)
presence of aspartate, glutamate, arginine, lysine, histodine and their
amounts.
d) pI
value or isoelectric point should be - pI > pH positive charge and pI >
pH negative charge
Effect of
pH:
Increase in pH caused by retention of acidic
groups by anions.
Decrease in pH caused by retention of basic
groups by cations.
The pH of the buffer must be between the pI
or pKa of the charged molecule and the pKa of ion-exchange resin.
Application:
1. used to convert one salt to another, for
example, tetra-propyl-ammonium hydroxide
from a tetra-propyl-salt of another anion.
2. useful for preconcentration of trace
components of a solution for analysis.
3. For the preparation of deionized water.
4. Separation of similar ions such as Na, H,
K ions by cation exchange and Cl, Br, iodide ions by anion exchange resin.
5. Measurement of active ingredients in
medical formulations.
6. Measurement of drugs and their metabolites
in serum and urine fir residue analysis.
Advantages:
a) easy detection of inorganic salts.
b) separation and purification of mixtures of
ions.
c) Long life of resins
d) Least expensive
e) Environment friendly
f) Controlled by pH, salt concentration,
ion-exchangers
g) A large volume of sample can be applied.
Disadvantages:
a) column efficiency is less
b) difficult to control over selectivity and
resolution
c) stability and repeated use of the column
may cause problems.
"Column Chromatography: Ion - exchange Chromatography: Types, Principle, Application, Advantages and Disadvantages"
Written By
Sadia
Akhtar
Student of
Department of Microbiology
Jagannath
University.
Email-
sadiabd810@yahoo.com
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