separation of threonine and glycine

[andrewn]

I am having trouble separating threonine and glycine (standards and in biological fluid) and wondered if anyone has experience of this separation?

I have tried Aqua C18, Develosil C30 and Ultratechsphere ODS columns with KH2PO4 Phase A methanol Phase B (95:5 to 30:70 over 25 min period). pH is 6.2. They elute at about 15 min.

Would changing the pH be my best option or trying to do something with the gradient?

Any help would be appreciated.

thanks
nick

[Kostas Petritis]

Nick,

Are you amino acids derivatized with something or underivatized (from the retention times you achive I guess they are derivatized so the question is with what)?

In the case they are not derivatize what is your detector?

[Steve]

Here is a procedure for the analysis of the two products.
https://www.macherey-nagel.ch/web/MN-WE ... =HPLC00017

It looks like you may have to alter temperture to get the best performance out of the columns.

[andrewn]

Kostas

my amino acids are derivatised with o-phthalaldehyde (OPA) and mercaptoethanol. As we have done this before for other amino acids such as glutamate and aspartate (which elute early and are easy to resolve).

nick

[andrewn]

thanks Steve. I didn't use the reference you supplied since what they called resolved peaks I would not! However, the suggestion you made about temperature did the trick! I reduced the temperature (from 40 to 25 degrees) and the two amino acids resolved from each other.

So thanks very much
nick

[Steve]

Glad to be of service. Good luck and hope to here from you again !

[Benjamin]

Dear Andrewn,

Even though you have resolved your problem, I would like to add the following for future problems.

OPA derivatives are not very stable, and some apirs such ALA/GLY are notoriously difficult to resolve by some column/mobile phase systems. Al alternate method that is likely to be better in some respects is to use FMOC derivatives instead.

The FMOC derivatives are better in terms of stability (1 day or so, instead of 1-2 hours for OPA). They are easier to separate and to my knowledge there are no significant resolution problems. However, some people object to the formation of the FMOC-OH byproduct. This however can be easily resolved from the peaks of interest.

Good Luck,

Benjamin

[Einar Ponten]

The most straightforward you can get...
.. is using ZIC®-pHILIC for this application.

A polymeric HILIC column designed for the most troublesome samples we have.