HPLC peaks

Debora Fontanini dfontani at facstaff.wisc.edu
Fri Jun 1 14:48:23 EST 2001

At 07:03 PM 6/1/01 +0000, Pablo Velasco wrote:
>Hi, all.
>I am starting to work in glucosinolate extraction. The first anaylisis in
>the HPLC were good with the peak that I need aroun 13m. But now, peaks
>appear around 7, 8 m. until I reset the HPLC three or four times. I am
>afraid that somethin else could be wrong.
>Does someone any idea about this?
>Thanks in advance,

Hi Pablo,

         I am sure many people in this forum can give you better advice 
than myself, but as far as I know, a few things could be going wrong in 
your separation.  However it would help to know what kind of method you are 
using; I am not sure what you mean by "reset".  Is that a 100% wash with 
your solvent B? baseline run? column regeneration?
         Are you sure your column is well equilibrated between runs? it 
usually takes about 10 column volumes to equilibrate back in buffer A, 
depending on what you end your gradient at. Are you washing with 100% at 
the end of each run? your column could get overloaded with strongly binding 
material, and not bind as well over many runs.  Are you using a guard 
column that might be old and losing its binding properties or somewhat 
interfere with the binding capacity of your column?  Or maybe is your 
column itself that is getting "bad" and you might try and regenerate it.

Just a few ideas,


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