Chromatography Forum

Hi all!

I'm trying to fine tune an HPLC method for FMOC-derived glyphosate, AMPA and glycine. I'm using tetraborate buffer (pH 9) for the derivatization, overnight at room temp, with FMOC-Cl being in sufficient excess as far as my calculations go.
The mobile phase is 5 mM ammonium acetate buffer/ACN, going from 25% ACN to 40% in 25 mins, then increasing the ACN a bit faster to elute residual FMOC, short flushing at 100% and equilibration back at 25%. The column is a Phenomenex 4.6x250 mm/5 um C18 operated at 40C (increased from 26C to improve RTs & peak shapes).

There are two issues I can't shake, would be grateful for your help:

1. Glycine derivatization seems to be more effective in a mixture of all three analytes vs. alone - as in, over 100% difference in peak areas. AMPA and glyphosate don't have this problem. Has anyone encountered this before?

2. The glyphosate peak (and to a lesser extent AMPA too) is tailing. Increasing the temp helped some but not substantially. I was thinking of trying 45C, are there any considerations against this if it's within column operating range? Any other ideas?

Detection is UV, FLD not easily available but possible if there's no other way.


Thanks! Any input would be greatly appreciated.

Have a look at the Agilent method, here

https://www.agilent.com/cs/library/slid ... 0Steed.pdf

Regards,
JMB

Thanks JMB! Could you be more specific as to what you're recommending in their method? There are some differences vs. what we use, but I'm not sure which of them is significant.

The most obvious difference is the column.
You use a generic Phenomenex C-18, while Agilent uses one of three different specific Eclipse columns.

If getting an Eclipse is not on the cards, then I suggest that you use their mobile phase and HPLC conditions with the Phenomenex column.

Since you have to resolve only 3 analyses, this approach may be sufficient.
If this does not resolve the analytes, then inspect the Agilent chromatograms to select one of the Eclipse columns for your analysis.

Regards,
JMB

Thanks! Will definitely try that.