5. Ion Exchange Chromatography Flashcards
What are the principles of ion exchange chromatography?
It sorts proteins based on their anionic/cationic strength where their accessible surface charge binds to the functional group of the matrix. Charges of proteins and functional groups are opposite.
What factor contributes to better binding?
Increased exposure of surface charge.
What does the buffer contain? What does it do?
Counterions. They bind weakly to the functional groups of the column and get displaced by the protein samples.
What are the types of ion exchangers?
Cation and anion.
What are the charges of the functional group and proteins they attract for the different types of ion exchangers?
Cation: -ve functional group; +ve proteins
Anion: +ve functional group; -ve proteins
What should you consider about the proteins when choosing type of ion exchanger?
The charge and stability of the proteins at different pH.
What should the charge of the buffering ions be?
They should be the same as that of the functional group
What should the buffer pKa be?
+/- 0.5pH units of desired working pH.
How do you elute bound proteins?
By increasing ionic strength using non-buffering salts like NaCl.
What is the mechanism behind counter ions and the elution of your proteins?
Eg. Cation exchanger: +ve counterions bind to -ve functional group. Add proteins. Proteins displace counterions and counterions flow through. Add non-buffering salt. Na+ displaces your bound proteins by binding to the functional group. Cl- binds to your displaced proteins to prevent rebinding to functional group.
Vice versa for anion exchanger.
What are the two elution strategies and when do you use them?
- Step elution: when you are familiar with your protein elution profile.
- Gradient elution: when you are not familiar with your protein elution profile.
What makes step elution better than gradient elution and vice versa?
Step > gradient cause faster and less buffer consumption.
Gradient > step cause better resolution of proteins.
What are the factors that affect resolution?
Flow rate: slower = better resolution.
Sample volume: lesser = better resolution.
What do you do to the elution buffer to allow proteins to elute?
Increase counterion concentration.
Can a protein of net charge 0 still bind to the functional group? Why?
Yes. As long as the surface charge of proteins are opposite of that of the functional group, they can still bind.
