General idea about ion-exchange chromatography

1. CENTRAL UNIVERSITY OF JAMMU.
Department of Chemistry and Chemical Sciences.
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Subject: Analytical Chemistry.
Teacher In charge: Dr. Shivender Saini.
Topic: Ion-exchange Chromatography.
Presented by: Zeeshan Nazir.
Roll Number: 3940319 (39).

2. Contents;
1. Introduction.
2. Types.
3. Principle.
4. Iso-electric point.
5. Procedure.
6. Salt based elution.
7. PH based elution.
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3. Introduction.
• Ion exchange chromatography is a process that allows the separation of ions and
polar molecules based on their charge.
For example: Proteins, nucleic acids etc.
• Mobile Phase is buffer solution.
• Stationary phase consists of a matrix containing charged ionizable functional
groups.
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4. Types.
• Cet-ionic Exchanger. An-ionic Exchanger.
• Beads Used: Resin quaternary ammonia Resin methyl sulfate
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5. Principle.
• Ion exchange chromatography separates molecules based on their
respective charged groups.
• A protein`s net surface charge changes with pH.
• At a particularly loading buffer pH, all appropriately charged proteins
will bind the resin.
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6. Iso-electric point ( pHi ).
 Iso-electric point of a protein is a pH where a particular protein molecule doesn’t contain
any charge or net charge is equal to zero.
 H OH
│ │
H₂N──C──C=O
│
R
1.) 2.) 3.) 4.)
H OH H O⁻ H OH H OH
│ │ │ │ │ │ │ │
⁺H₃N──C──C=O H₂N──C──C=O H₂N──C──C=O H₂N──C──C=O
│ │ │ │
R R R⁺ R⁻
 (+ive Charge ←) Lower pH < pHi < Higher pH (→ -ive charge)
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7. • A mixture of 3 proteins was provided.
• pHi = 3 pHi = 7 pHi = 9
• Buffer used has a pH = 5.
• Charge attained by each protein molecule;
A= -2 B= +2 C= +4
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A B C

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9. Salt based elution.
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10. 10

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12. PH based elution.
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13. Buffer solutions of PH 8 and PH 10 will be used now in a
respective manner for protein B and protein C.
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16. THANK YOU SO MUCH!
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