1. Ion exchange chromatography

2. What is ion exchange chromatography ?
IEC is a technique for separating proteins according to their charge
Its easy of use and scale up capabilities
Large volumes be applied
High resolution and high capacity method

3. Principle Packing material Resin are charged molecules
Securely bound to column by covalent bonds
Negatively or positively charged groups
Anion exchanger
Positively charged beads “exchange” with negatively charged counter ions
Negatively charged molecules - “anions”
Cation exchangers
Negatively charged beads exchange positive counter-ions (cations)

5. Functional Group
Counter-ion
Anion Exchanger
Diethylaminoethyl (DEAE)
-O-CH2-CH2-N + H (CH2CH3)2
Cl -
Cation Exchanger
Carboxymethyl (CM)
 -O-CH2-COO -
Na +

6. Separation of protein Proteins are charged molecules
Interaction with the packing material depends on
Overall charge
Distribution of that charge over the protein surface
They displace mobile counter ions bound to the resin
Mobile counter ions: When the packing material is suspended in buffer containing NaCl, the charged groups become loosely associated with Na+ and Cl-ions of the opposite charge. These loosely bound ions are called mobile counter-ions.

7. Net charge on protein will depend on
Composition of amino acids in the protein
pH of the buffering solution.
Charge distribution will depend on
How the charges are distributed on the folded protein
Isoelectric point ( pI) - The pH at which a particular protein is overall electrically neutral ie. the number of positive charges is equal to the number of negative charges.
< pI - A protein has more positively charged amino acids and therefore an overall positive charge. It will bind to cation exchangers
> pI - A a protein has more negatively charged amino acids and an overall negative charge. It will bind to anion exchangers
At its pI, a protein will not bind to either a cationic or an anionic exchanger.

8. Column materials - Polystyrene
Ion-exchangers made by co-polymerisation of styrene with divinyl benzene. Polystyrene itself is a linear polymer.
Divinyl benzene, is a cross-linker
Resins with low degree of cross-linking are more permeable to high molecular weight compounds, but they are less rigid and swell more when placed in buffer
Sulphonation of cross-linked polystyrene results in sulphonated polystyrene resin such as Dowex 50- strong acidic exchanger
Basic exchangers are prepared by reacting cross-linked polystyrene with chlormethyl ether and then by the chlorogroup with tertiary amines – CH2 N+ (CH3)3 Cl- groups are ionized.

9. Modified cellulose and Sepharose
Modified cellulose is an alternative to polystyrene based exchangers
Cellulose is a high molecular weight i.e. carboxymethyl cellulose (CM-cellulose) where the – CH2OH group is converted to –CH2OCH2COOH and DEAE-cellulose [CH2OCH2CH2N(CH2CH3)2]
Commercially available in gel and bead form
Sepharose type is derived from cross-linked agarose
Sephadex and Sepharose are used for the separation of high molecular weight proteins and nucleic acids

10. Total exchange capacity
Defined as number of milliequivalents of exchangeable ions available either per gram of dried exchanger or per unit volume of hydrated resins
Bio Rad AG1-X4 = 1.2 meq cm-3
DEAE-Sephadex A-25 = 0.5 meq cm-3
CM-Sepharose CL-6B = 0.2 meq cm-3
Polystyrene exchangers are obtainable in a number of mesh size. All exchangers are supplied with counter ions i.e Na+ or Cl-.

11. Classification of ion exchange media
Media (X = matrix) 
Nature 
pH range 
Applications 
Anion exchangers 
X-CH2N+(CH3)3 
strong 
2 - 11 
nucleotides 
X-CH2NH+(CH3)2 
intermediate 
2 - 7 
organic acids 
(CH3CH2)2 X-CH2CH2NH+ diethylamino-ethyl (DEAE) 
weak 
3 - 6 
proteins 
Cation exchangers 
X-SO3- 
amino acids 
X-COO- 
6 - 10 
peptides 
X-CH2COO- carboxymethyl (CM) 
7 - 10 

12. Buffer
The pH of the buffer used should be one pH unit above or below isoionic point of the compounds.
Cationic buffers are tris, pyridine and alkyl amines and they are used with anion exchangers.
Anionic buffers are acetate, barbiturate and phosphate and they are used with cationic exchangers.

13. Sample application
Amount dependent upon size of the column and capacity of exchanger.
For the isocratic elution, sample volume is 1-5% of bed volume.
For the gradient elution, sample volume is not important.
Large volumes of dilute solution can be applied as they get bound at the top of column.

14. Elution Gradient elution is most common and gives better results.
With anion exchanger, pH gradient decreases and ionic strength increases.
With cation exchangers, both pH and ionic gradients increase.

15. Applications
Separation of amino acids achieved by strong acid cation exchanger – Dowex 50-PS and SP-Sephadex-cellulose
Separation of proteins by weakly acidic and basic exchangers derived from cellulose and agarose
Proteins with isoionic point < 7 – DEAE-cellulose using low ionic strength
Proteins with isoionic point > 7 – CM-cellulose using buffer pH 4-5
Proteins with isoionic point  7 – Either
Determination of base composition of nucleic acids
RNA hydrolysed by both enzymatic and base hydrolysis. DNA resisted to base hydrolysis but cleaved by DNAases.

16. Separation of amino acids achieved by strong acid cation exchanger – Dowex 50-PS and SP-Sephadex-cellulose
Introduction of sample at pH 1-2 to ensure complete binding
Gradient elution by increasing pH and ionic concentration
Acidic amino acids, aspartic and glutamic acids elute first followed by neutral amino acids, glycine and valine. Basic amino acids retain their net negative charge up to pH values 6-11 and elute last.