I am currently working on glyphosate derivatization with FMOC and having a trouble to get recovery of the spiked sample. I am using grain Metrix specially, Barley, wheat and oats. listed below my project steps:
Extracted sample from pluses, then Take 0.5 ml sample + adjust pH 9-10 + add 250 ul Borate buffer + 250 ul fmoc the after 30 min add 60 ul 98% FA to stop derivatization.
Not sure where am I doing wrong but still not have enough recovery. suggestions much appreciated! thanks!
It has been a long time since I worked with FMOC derivatizations. Wheal et al 2002
Article Reversed-phase liquid chromatographic determination of phyto...
Your method seems to have a number of ingredients and requirements to it. e.g. you adjust pH to 9-10, then add Borate buffer THEN add the FMOC, for a set time (30 min.).
Have you tried simplifying it to see where you can get maximum recovery from a blank, then adding matrix to determine what might be decreasing your yield?
- Have you checked that pH 9 is optimal?
- Can you combine pH and Borate buffer? (*What is the borate buffering, is it needed?)
- FMOC presumably breaks down over time. Are you sure FA (whatever that is) is not breaking it down faster than you can measure the derivatives?
- Thinking of timing - how quickly do all these liquid handling steps take: seconds between samples, or minutes?
- I would try a few (zero, low, medium, high) glyphosate concentrations and see if you get better recovery with some adjustments over others.
- Once you can get a good recovery, see how much mass of sample you can add before you start to see under-recovery. Maybe there is something in the carbohydrates than binds glyphosate or breaks it down before the FMOC even gets to react.
Thanks Matthew!
I checked that the pH9 is optimal for glyphosate binding. And for rest I will take an account and will do the experiments again. Thanks A lot!
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