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Elution questions
Ryan_5248
#1 Posted : Monday, June 07, 2021 11:20:59 PM
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Hi, I was a bit confused about elutions during chromotography/separation.

I wanted to know how you elute something from chromatography? I heard in class that it's always done with high salt concentrations.

However, during my Altius FL#1, a standalone question asked:

Which change in solution composition would cause a protein to elute from a hydrophobic interaction column?
The correct answer was a low salt concentration.

I'm kind of confused on the science behind elutions. How do they work? Is there a table that I should know.


Thanks!
INSTR_Molly_129
#2 Posted : Saturday, June 19, 2021 7:13:05 PM
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Hey Ryan, great question.

With chromatography, it will depend on the type of column that you are using AND the type of solvent that is being pushed through.

With a hydrophobic interaction column, it means that its super duper greasy.

There are two types of solvents being used in chromatography as well, a polar and a non-polar solvent, each at different mixture ratio to make 100% of the solvent passing through the column. When you run chromatography, it can be ie. 20% polar + 80% non-polar solvent (These numbers are NOT something you need to remember, I'm just giving an example). The changing gradient of the polar vs. non-polar phase is what pushes materials out, based on its polarity.

The polar phase is the "salty" phase. So when the answer is saying "low salt concentration", its talking like for example, 10% polar solvent, 90% non-polar/greasy solvent. And so with very high concentration of greasy solvents running through the very greasy column, your materials will no longer only want to bind to the greasy column, but will want to bind to ALL of the greasy solvent too, making it easy to elute, or move to the ejection end.

I hope that helps, Ryan. Let me know if you have any more questions.

Happy studying,

Molly
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