[phth]

Is there a way to prove if a compound is decomposing by HPLC like a 2D TLC plate? Other than isolating a peak, weighing, re-isolation. Thx

[Babcock_Hall]

I might well be misunderstanding your question.  However, I was once able to see a change in retention time of a protein that had been biotinylated with a maleimide-based reagent.  If I am not mistaken the ring is hydrolytically labile, and I tentatively ascribed the change in retention time to this reaction.  Therefore, I would say that a change in retention time is indicative of decomposition but the lack of a change in retention time is less easy to interpret.

[rolnor]

LC-MS is great if you have it. You could get some information from TLC, it depends on your compound, can you give the structure?

[TheUnassuming]

I'd take the most concentrated fraction off the HPLC and run UPLC or LCMS of the fraction over time to see if its degrading.  In theory, unlike normal phase the reverse phase column/HPLC itself shouldn't be doing anything to your compound.  So its just a matter of how stable your product is in your mobile phase. 

Random side question.  How are you concentrating your HPLC fractions?  In my current lab, most people rotovap them down because its faster, but if your compound has has any hydrolytic instability you will see decomp and need to lyophilize instead.

[phth]

Thanks for the replies, but no info on the structure other than it's hydrolytically unstable. Isolation by dilution to wash and lyophilizaiton. I isolate the major peak by RPHPLC and it elutes as one peak (or more in another case). Upon identical reinjection by analytical RPHPLC, it decomposes to multiple discrete peaks. What I don't understand is that if it decomposes it should be a gradual event smearing the compound, and not discrete peaks...

[MOTOBALL]

I think that multiple, clean, discrete peaks denotes hydrolysis in the vial.
Decomposition actually occurring during the elution would result in the smeared, major peak that you describe.

Regards,
Motoball