Column Chromatography: Ion - exchange Chromatography: Types, Principle, Application, Advantages and Disadvantages

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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