Chromatography Forum

Hi,

I need help. Does anybody have some experience with chiral separation of N-acetylserine or 3-aminopropane-1,2-diol in HPLC??

Both molecules are quite small and it´s problem with detection, because 3-aminopropane-1,2-diol doesn´t have any chromophore and N-acetylserine has only two carboxy groups.
I tried pre-column derivatization of 3-aminopropane-1,2-diol with OPA reagent. Problem with detection was solved, but separation didn´t pass.
Then I tried post-column derivatization and for separation used Crownpak column, which is for separation of amino groups, but racemic mixture wasn´t separated.
I´ve tried it separate also on capillary electrophoresis after derivatization with dansyl chloride, but separation wasn´t good.
I will be very glad for every advice.

Thanx

Peter

This should be pretty straightforward. Just OPA derivatize both and you should be able to separate the diastereomers formed using a good reversed phase HPLC column. Try it.

How does OPA form diastereomers?

Derivatization with an enantiomerically pure substance could be the way to go, though, but:
unless you have only extremely low amounts of the amine I don´t understand that you have a UV detection problem.

To separate chiral amines, derivatise with Marfey's reagent and run on a C18 column.

Peter

Of coures you will need a chiral OPA reagent. It needs to be the thiol. I like to use N-Acetyl-S-Cysteine which then produces sets of diastereomers which can be separated by reversed phase HPLC. Works great!

Amazing! What sort of absolute yields did you get? I junked the conventional OPA method (used just for fluo. detection with the standard thiol), because it was messy.