Chromatography Forum

Hello,

I am attempting to analyse the 20 amino acid standards on a 30m DB-5 column.
I am using 1ml BSTFA + 1%TMCS, 1ml pyridine and heating at approx 80-85 degrees for 1 hour. I am getting peaks for all the derivatives(though some almost co-elute). However my problem is that for four of the amino acid standards I am obtaining two peaks as follows:

Aspartic acid 6.2 mins and 9.75 mins

Glutamic Acid 11.4 mins and 13.36 mins

Asparagine 11.66 mins and 12.23 mins

Glutamine 13.36 mins and 13.87 mins

Would anyone have a suggestion as to what is happening with these four amino acids?

Thanks.

GCMS (if you have it) can tell you whether you've fully derivatized all the -N-H sites; the C-O-H sites derivatize much easier.

Hello,

Thanks for the reply. Unfortunately I don't have GC-MS. However I will try higher temp/longer derivatization time to see if any or all amino acids reduce to one peak.

Thanks.

I'm afraid longer derivatization won't help with BSTFA. If you use MSTFA or MTBSTFA, you will find most of the aa's you've listed will elute as single peaks due to more complete derivatization; however, glutamine and asparagine will still give double peaks. If you increase heat or derivatization time, glutamine and asparagine will break down to glutamate and aspartate, respectively.

Put initial oven temp higher