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Chromatography Elution

Question: Sketch the expected chromatographic elution profile (protein concentration vs elution volume) if a mixture of these proteins is applied to a weak anion exchange resin such as DEAE cellulose at a pH of 7. Label and show the salt gradient used for elution.
Protein:
(A), pI 8, MW 25000
(B) , pI 10, MW 10000
(C) , pI 4 , MW 70000
(D) , pI 6, MW 5000

Solution Preview

Instead of sketching the graph, let's look at the concepts involved. That way, once you understand what's going on, you should be able to sketch the graphic easily yourself.

First concept:
Ion exchange resins contain charged groups. In the case of an anion exchanger such as DEAE-cellulose, the charged groups are basic. On the other hand, for a cation exchanger such as CM-cellulose, the charged groups are acidic in nature.

Additionally, cation and anion exchangers may be assessed into weak and strong exchangers depending on how well they bind the counter ions.

Second concept:
Protein samples are loaded under onto an ion exchange column at low ionic strength. This helps the proteins to bind to the column because low ionic strength promotes electrostatic interactions. Therefore, once the proteins are bound to the column, they are eluted using either a step or gradient elution of buffer with a higher ionic strength. Higher ionic strength promotes dissociation of ionic interactions and so helps facilitate the proteins coming off the ...

Solution Summary

This solution provides a detailed explanation of the concepts which relate to this topic so that a student can really grasp the subject well. A description of how to sketch the profile is also given. This solution is a great study tool for understanding this specific subject material.

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