aminoacid hydrolysis and HPLC analysis

[hplcnewuser1]

Hi everyone, my product is a peptide 14aa long and I need to hydrolyze and show aminoacid content. Does anyone have an idea how to go about it as far as sample prep and HPLC method for aminoacid separation.
Any help is appreciated.

[HPLC chemist]

See this thread and sigmaaldrich has a website that may help.

https://www.researchgate.net/post/Deriv ... 0d394763c3

[Blazer]

The USP has a monograph on amino acid analysis. That can be a place for you to start. I also recommend looking at the Waters or Agilent websites. Both offer kits and application notes for this. I've not used the Agilent ones, but have had great success with the Waters ones in the past.

[hplcnewuser1]

Thanks for that info I will check on it.
My question is a bit more specific. I will use 6M HCL with 20%tfa in 110C for 72 hours. What sort of glass tube can withstand these condition. I do not need nitrogen because there is no tryptophan. Also is vacuum necessary for a pwptide hydrolysis that has only I K and E? How can we set up in a safe way to mke sure the glass container does not crack or explode.

[qfiviera]

I had hydrolised collagen with 6 M HCl for 24 hours at 110 °C in a dry bath (alluminium blocks) in restek head-space vials (GC) and never had any problems. For LC used Hypersil ODS AAA from agilent, 5 µm, 200 x 2.1 mm, gradient mode. Derivaized with FMOC chloride.

[hplcnewuser1]

actually i tried pierce tube with 1mL of 6M HCL and some peptide and phenol as well. my question is how long should it be vacuumed for? I tried vacuum 5sec and the acid boiled up the tube when temperature reached 70C in heating block. Is this normal? Is it a vacuum issue or something else.
What volume vials did you use. If I use 1ml sample and acid is a 2ml gc vial ok?

Thanks

[hplcnewuser1]

My peptide is not soluble in 6M HCl. Does the peptide need to be soluble in acid in order to do acid hydrolysis?
Thanks