Answer to Question #109222 in Biochemistry for kylie

Question #109222
Discuss the principles of ion exchange, affinity and gel exclusion chromatography
1
Expert's answer
2020-04-22T08:07:59-0400

Introduction

Chromatography is a laboratory technique that is used to separate mixtures based on their interaction between the mobile phase- where the mixture to be separated is placed and the stationary phase.


Principles.

Ion exchange chromatography

The ion-exchange chromatography is a chromatography technique that separates molecules based on their charge or polarity. The stationary phase of this chromatography consists of agarose. A molecule of the opposite charge to the molecule of interest is always immobilized on the agarose. When cations are separated, then the system will be called cation exchange chromatography. The anion exchange chromatography separates anions.


Affinity chromatography

Affinity chromatography is a separation technique based on the specific interaction between an immobilized ligand, and it's binding protein. For example, nickel which has a high affinity to histidine, can be used to separate histidine from a mixture of other protein. Usually, the mixture to be separated is injected into the stationary column and allowed to flow. Those molecules with high affinity to the ligand on the stationary column will move slow and be eluted last unlike those with low affinity.


Gel exclusion chromatography

The gel exclusion chromatography is also called the size-exclusion chromatography. In this separation technique, molecules are separated on the basis of their size as they move through the stationary phase. The stationary phase is made of agarose, which has minute pores. Large particles, bigger than the pore size of the gel, cannot enter the gel and thus they flow faster and get eluted first. On the other hand, smaller particles can enter the pore of the gel and therefore take a lot of time to transverse the gel volume and thus eluted last.




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