Scribd
Upload
14 views18 pages

Unit 3 1

Ion-exchange chromatography is a technique used to purify polar proteins by separating them based on their net charge at a specific pH. It involves cation and anion exchangers, which utilize different functional groups for binding proteins, and employs salt and pH gradients for elution. Common applications include water softening, demineralization, and the separation of charged molecules.

Uploaded by

56x62tkmpn
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
14 views18 pages

Unit 3 1

Ion-exchange chromatography is a technique used to purify polar proteins by separating them based on their net charge at a specific pH. It involves cation and anion exchangers, which utilize different functional groups for binding proteins, and employs salt and pH gradients for elution. Common applications include water softening, demineralization, and the separation of charged molecules.

Uploaded by

56x62tkmpn
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
You are on page 1/ 18

Ion-Exchange

Chromatography
Introduction
• Ion-exchange chromatography purify the
POLAR proteins
• Three types of separation may be achieved:
(1) ionic from non-ionic,
(2) cationic from anionic, and
(3) mixtures of similarly charged species.
Principle
• Separation based upon differences in their net
charge at a particular pH.
• Protein charge depends on the number and
type of ionizable amino acid side chain groups.
• Each protein has an isoelectric point (pI)
bind to the negatively
Protein is positively charged functional
Below pI
charged groups of a cation
exchange resin

Binds to positively
Protein is negatively charged functional
Above pI
charged groups of an anion
exchange resin
• Separation also depends upon charge of an analyte:

Anionic
exchanger

NOTE: M2- BINDS TIGHTLY AS COMPARED TO M- AND HENCE ELUTED AT THE LAST

https://onlinecourses.nptel.ac.in/noc19_bt15/preview
Types of ion exchanger
• Cation exchanger (acidic ion
exchangers)
• anion exchanger (basic ion
exchangers)
Anion exchangers Functional group
-
diethylaminoethyl (DEAE)-
OCH2CH2N+H(CH2CH3)
cellulose
2
Triethylaminoethyl-dextran -OCH2CH2N+(C2H5)3
Trimethylaminomethyl-
-CH2N+(CH3)3
cellulose
Cation exchangers Functional group
carboxymethyl (CM)-
-OCH2COO-
cellulose
sulphopropyl (SP)-
-CH2CH2CH2SO3-
polystyrene
-
https://onlinecourses.nptel.ac.in/noc19_bt15/preview
Materials
• Matrices used include polystyrene,
cellulose and agarose

• Strong exchanger = totally ionised at all


normal working pH values
• e.g.–SO3– (sulphonic acid moities) and –
N+R3 (quaternary amine groups)
Weak exchangers = ionised over only a
narrow range of pH
• e.g. –COO- and diethylammonium (–
HN+(CH2CH3)2)
Elution
1) A salt gradient is used to elute separated
proteins.
2) A decreasing pH gradient
elute proteins from an anion exchange resin
3) an increasing pH gradient
1) elute proteins from cation exchange resins.

Decreasing the pH of the


Increasing the pH of the

Protein becomes less Protein becomes more


protonated
mobile phase
protonated
mobile phase

Can’t form an ionic Can’t form an ionic


interaction with negatively interaction with positively
charged resin charged resin

Hence eluted Hence eluted


OPERATION OF THE TECHNIQUE
Same steps as for the other two
1. Column packing
2. Sample preparation and loading
3. Elution
4. Column Regeneration
APPLICATIONS
1- Water softening:
Removal of ions cau sing hardness of water like Ca and Mg
2-Water demineralization
Removal of cations & anions dissolved in water.
3. Separation of electrolytes from non-electrolytes.
4-Separation and purification of charged molecules
e.g. Uronic acids separated on anion exchanger.
Hexosamines separated on strong cation
exchanger.
4-for the separation and purification of
charged molecules
e.g. Uronic acids separated on anion
exchanger.
Hexosamines separated on strong
cation exchanger.
Pollinq
Ion exchangers are used for the
separation of
A) Polar molecules
B) Non polar molecules
c) Both of the above
 To elute proteins using an anion exchange resin,
a) Decreasing ph gradient is used
b) Increasing ph gradient is used
 Ion exchange chromatography is based upon
a) Partition chromatography
b) Electrostatic attraction
c) Adsoprtion chromatography
d) Mobility of ionic species
POLLING QUESTION

 CATION ECHANGERS HAVE


A) ACIDIC FUNCTIONAL GROUPS
B) BASIC FUNCTIONAL GROUPS
Video links

 https://www.youtube.com/watch?v=VOSkyj1dtbc
 https://www.youtube.com/watch?v=i4U4ndf2ayg –
good

You might also like