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Chemistry Forums for Students => Organic Chemistry Forum => Organic Chemistry Forum for Graduate Students and Professionals => Topic started by: phth on June 26, 2019, 01:02:19 AM

Title: HPLC decomp
Post by: phth on June 26, 2019, 01:02:19 AM

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

Title: Re: HPLC decomp
Post by: Babcock_Hall on June 26, 2019, 10:26:22 AM

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.

Title: Re: HPLC decomp
Post by: rolnor on June 26, 2019, 11:58:19 AM

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?

Title: Re: HPLC decomp
Post by: TheUnassuming on June 26, 2019, 06:55:23 PM

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.

Title: Re: HPLC decomp
Post by: phth on June 27, 2019, 02:25:33 AM

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...

Title: Re: HPLC decomp
Post by: MOTOBALL on June 27, 2019, 03:43:59 PM

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