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Internal standard for HPLC - (Jun/28/2011 )

Hi all,

I am trying to find a new internal standard for my HPLC method as the old IS seems to be unstable. Everything I have read indicates that the IS should elute AFTER the compound of interest. However, most of the chemically similar compounds to my compound of interest will elute BEFORE my compound. Is it a problem if the IS elutes before the compound of interest? Or is it just important that it elutes near to the compound of interest, without the peaks interfering with eachother?

Thoughts are appreciated.




After is definatly best but not always achievable. What are your reasons for having IS? And are you going to be using IS for calculations/data adjustments or just to help with "eye-balling" the data?

What are your elution gradient parametres and can they be changed? If they can then you will have a lot more options.


Hello :)

what's important is that the IS has a similar chromatographic retention time to your analyte of interest.
this what i know from my experience ...

some references will say it's better to have it eluting first, others will say no ...
BUT, all agree that it should have a similar elution time.

so, if i were you ... i wouldn't worry.