ion exchange chromatography - ksufac.ksu.edu.sa/sites/default/files/bch_332_lecture_11.pdf•ion...

Ion Exchange Chromatography

BCH 332 lecture 11

1

• Ion exchange chromatography is a form of adsorption chromatography in which charged proteins or other biomolecules are exchanged for small ions of like charge originating in salts (e.g. Na+, Cl−).

• These ions are attached to chemical structures on the surface of the stationary phase called ion exchange groups or ion exchangers.

2

• Two types of ion exchanger are common in biochemistry;

• Anion exchangers (which exchange negatively charged ions or anions) and

• Cation exchangers (which exchange positively charged ions or cations).

3

4

5

6

• Some ion exchangers are regarded as weak, that is functioning best over a comparatively narrow pH range, while others are strong, that is functioning over a wider pH range. Exchangers with quaternary amino or sulfonic acid groups behave as strong anion and cationexchangers, respectively, while aromatic/aliphatic amino and carboxylic acid groups are weak anion and cation exchangers, respectively.

• Choice of ion exchange stationary phase is heavily influenced by knowledge of the pI of the protein of interest.

7

8

• Desalting is carried out before ion exchange either by gel filtration chromatography , by dialysis or by centrifugal filtration.

• Elution of bound proteins is achieved by reversing the process of binding and, again, exchanging a counterion for protein.

• This is usually carried out by applying a large excess of a salt (e.g. NaCl) containing the counterion in the mobile phase.

• Because proteins have different net charge, they may bind to an ion exchanger at a given pH with a variety of strengths, that is some proteins may bind strongly whilst others bind weakly or not at all.

• We can take advantage of this to separate proteins by applying salt in a continuous gradient. Weakly bound proteins will elute first from such a system while strongly bound proteins elute later.

9

• Mobile phase Acids, alkalis, buffer.

• Stationary phase The ionic compound consisting of the cationic species (M+) and the anionic species (B-)

• Elution - Components of mixture separate & move down the column at different rates depending upon the affinity of the ion for ion exchanger. The eluates are collected at different stages

• Analysis of the eluate (Spectrophotometric)

10

11

12

Selection of an Ion Exchanger

13

Selection of an Ion Exchanger

14

15

• Below the isoelectric pH the protein has a positive charge and binds to a cation exchanger.

• Above the isoelectric pH the protein has a negative charge and binds to an anion exchanger.

16

Choice of Buffer

• Cationic buffer should be used with anion exchanger.

• Anionic buffer should be used with cation exchanger.

17

• The basic process of chromatography using ion exchange can be represented in 4 steps:

1. Equilibration

2. Sample application and wash

3. Elution

4. Regeneration

18

19

20

21

22

APPLICATIONS

• For extraction of enzymes from tissues.

• Separation of sugars, amino acids and proteins.

• Ion exchange column in HPLC.

23