Chromatography Forum

Hello!
I'm new to this forum, and I'm also new to chromatography (molecular biologist by education), so please HELP!
I'd like to separate and quantify few amino acids from plant extracts, in particular glutamate and 2 forms of oxidized methionine - methionine sulfoxide & methionine sulfone. I've been trying to do that by derivatization of amino acids with FMOC-Cl and then running the samples on C-18 column with citrate pH6.4 & methanol as solvents. Now my standards are fine, but there is a problem with samples, because of all other things present in plant extracts. I cannot see my peaks because there is so many other peaks. What would be the best way to purify amino acids before HPLC? I was thinking to use some SPE columns, but i'm not sure which type would be the best? I would greatly appreciate any suggestion or protocols. Thanx!

MostlyHarmless

Since the amino acid experts don´t take this:
This type of analysis seems to be among the toughest for LC. If your amino acids are at a relatively very low concentration it probably approaches impossibility.
You should at least get rid of the proteins and peptides (discussed many times: SPE, solvent extraction, ultrafiltration, dialysis, precipitation). What did you do so far?
Also you should know that FMOC gives a peak with H2O and NH3 (seemingly always present), also, it´s prudent to get rid of excess FMOC-Cl with either a highly polar amino acid or with adamantyl amine.
Furthermore, you might need a multidimensional (mullti-step) chromatography even after a rigorous cleanup.

I would suggest an anion exchange SPE clean up. Your compounds although zwiterionic they can be easily charged negatively so I would modulate accordingly the pH in order to retain my compounds and get rid of most of the matrix. I would gues that even with a pH 4 all your compounds are negatively charged (unlike most of the other amino acids, peptides and proteins...

As Hans said not very easy task, mainly because of the luck of specificity... (i.e. it would be much easier to do by MS)...

Thank you for the suggestions. However (thanx God) i think i've solved the problem in the meantime.... since i'm interested only in few polar amino acids, it looks like that SPE C-18 does a good job by binding everything else, so finally i got my peaks Thank you anyway!
MH