Derivatisation HELP needed

[just me]

hello
I am trying to derivatise 1mcg and less of amikacin by heating it at 85 degrees for 45mins in tris buffer pH11-12 with fluorodinitrobenzene and the cooling in the mixture in 4 degree fridge for 10mins afterwards . Sometimes it works and i get a decent calibration curve however more often than not it doesnt seem to work and i see nothing which means that it isnt passing the validation requirements.

Does anyone have any idea what could be going wrong ???

[HW Mueller]

Only a general point: Derivatizations of this type are extremely dependent on conditions, try to see whether you can learn to keep things constant.

[Mark Tracy]

I notice a few oddities in this procedure. First the pKa of TRIS is 8.5 not 11.5, so it is not really a buffer. Second, TRIS is a primary amine and so is your amikacin. You probably have competition between the two for the derivatizing reagent, and sometimes the amikacin loses the race. In fact, TRIS would be something you would use to eliminate excess reagent. I would switch to triethylamine.

[Patrik Appelblad]

Another general remark is that in many published studies, you will see that the molar ratio between the target compound(s), the catalyst and the reagent is completele unproportional. In fact, in my opinion I believe that there are several examples where it just have been an addition of catalyst and reagent purely by serendipity. Quite often this works, especially when derivatizing large amounts, however when going down in concentrations, the problems start to appear.

A few years ago I applied a chemometrical approach to pre-column derivatization of ketosteroids. I started of with a well known system, and in the end I was able to decrease the molar ratio between by orders of magnitude. I also changed the catalyst and the solvent, which all in all made it possible to decrease the reaction time dramatically. It also gave better control of the complete procedure, improving the reproducibility, yielding better LOD's.

I also agree with Marks remark, and perhaps there might also be other parameters worth checking up.

[just me]

hello

Thanks for you comments. I will try the trieythylamine however i am using a reversed phase column so hopefully the tris buffer pH7 will prevent any detrimental effects to it