Ion Exchange Chromatography Lab report write up
Set up the apparatus required and follow the general workflow associated with liquid chromatography
Detail the concept behind compound separation by size exclusion
Introduction:
Ion exchange chromatography is a powerful tool used for the separation and purification of macromolecules such as proteins or nucleic acids. The fundamental principle behind the technique is based on the interactions of the net charge of a molecule with the charges on the resin. In this report, we will discuss the setup protocol for conducting an ion exchange chromatography lab experiment along with a brief on the concept behind compound separation by size exclusion.
Set up of Apparatus:
The apparatus set up for ion exchange chromatography has the following components:
1) A low-pressure chromatography system
2) A column that is packed with the chosen cation or anion exchange resin
3) Buffers that have the same pH as the resin
4) The sample containing the target molecule
5) Fraction collector
The General Workflow of Ion Exchange Chromatography:
The general workflow of ion exchange chromatography is as follows:
1) Column equilibration: To prepare the column, it must be washed with the same buffer as that of the resin to create a homogenous packing. This step ensures better separation and purification.
2) Sample loading: Once the column is prepared, the target molecules need to be loaded to it. The samples are added to the column received by the resin and pass through the resin bed.
3) Washing: During this step, the unbound molecules are removed from the resin bed, leaving the bound molecules attached to the resin.
4) Elution: Elution is the process where bound molecules are removed from the resin by the buffer containing ions of higher affinity than the target molecule. These buffers with different ionic strengths or pH are used to elute the target molecule from the resin.
5) Collection: The sample fractions eluted from the resin via the elution process can be collected and stored.
Compound Separation by Size Exclusion:
Size exclusion chromatography is a technique used for the separation of compounds based on differences in size. This technique relies on the exclusion of molecules by the pre-packed resin bed. The resin bed has beads with a specific porosity that allows the passage of small molecules and excludes the larger ones. The smaller molecules will diffuse through the resin beads and take longer to elute, making it possible to separate them from larger molecules that elute earlier. This technique is useful in separating and purifying polymer molecules based on molecular size.
Conclusion:
Ion exchange chromatography is a powerful tool used for the separation and purification of macromolecules. The separation is based on the interactions of the net charge of a molecule with the charges on the resin. The size exclusion principle is useful in separating and purifying polymer molecules based on molecular size. The procedure outlined in the above report can be applied to separate and purify proteins, DNA, and RNA in a typical research laboratory.