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Question regarding HPLC


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Hi

 

Hypothetically speaking, if I run two compounds on an HPLC and they have poor separation/poor baseline resolution how can I improve the separation in terms of changing flow rate.

 

Do I increase the flow rate or lower the flow rate?

 

I know that lowering the flow rate will make the peaks wider.

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The unhelpful but accurate answer is that it depends.

For both the compounds the resolution will depend on flow rate in agreement with this sort of thing

http://en.wikipedia.org/wiki/File:Van-deemter.jpg

Without knowing a whole lot of other things about the situation it isn't possible to know where you are on that plot, so it's not possible to know if you would be better moving to the right (faster) or the left (slower).

Realistically, the only way to tell is to try it.

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  • 1 month later...

If the two compounds both elute early then they have a low capacity factor (k'), and possibly slowing the flow rate may improve separation up to a calculated k' of about 20 and after that the peak widening you mention will be pretty severe.

 

The best way and easiest way to improve selectivity of two closely eluting compounds is to change the chemistry of the system by (most easily) change mobile phase (eg. change from acetonitrile to tetrahydrofuran, add salts or adjust pH if compounds are pH sensitive). If that doesn't work, think of changing the stationary phase (eg. C18 column to amine column), and if that doesn't work perhaps there is a way to chemically derivatize one or both of the compounds to achieve better separation (eg. methylation, acetylation, etc.)

Edited by Carvone
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