Hi Brian,


Luckily we are in a position where we don't have to rely on projections and estimates but can carry out  a few simple experiments to provide us with some information.

If you have the time, run the following experiment (looking at 13C only).

Derivatize L-leucine as N-acetyl, O-n-propylate (NAP).

Derivatize L-leucine as N-TFA, O-n-propylate.

The main thing is to keep everything consistent, i.e. the leucine comes from the same pot and the derivatization agent for the carboxyl group comes from the same batch. Both derivatives contain the same number of carbon;  1 molequivalent of derivatized leucine contains 11 molequivalents of carbon.

Prepare a stock solution for each of the same concentration. I used 1 nmol/microL and injected as much (1 uL) on column with the split closed during the injection period.

Since this is a single compound experiment it doesn't matter what column one happens to have installed at the time. I agree, capillary thick-film columns deliver great peak shape and separation (I like especially a combination of 0.25 mm ID x 1 um). They are also more tolerant to multicomponent samples with a wide range of individual concentrations.

1. On a fresh (new) oxidation reactor (CuO/Pt only) inject 1 uL of the NAP derivative and make a note of the total peak area (or just the m/z 44 peak area), which represents (theoretically) 11 nmol of CO2.

2. Follow this up by an injection of 1 uL of the TFAP derivative and compare the peak area number you get here with the above. All things being equal and with no detrimental effects from fluoride poisoning the peak area numbers should matchup.

3. Repeat steps 1 and 2 a few times in an alternating fashion and keep monitoring peak areas.

If you have some more time repeat the above longitudinally for each derivative on dedicated fresh oxidation reactors (i.e. one a fresh reactor run NAP-Leu 10 times; swap the reactor for a fresh one and run TFAP-Leu 10 times).

Let me know what you find and how happy you are to accept the condition is met that the carbon isotopic composition of the CO2 produced (for the TAFP-Leu or any subsequently run compound) is a true representation of its parent material if we agree that only reactions proceeding in a quantitative fashion are isotopic fractionation free?


I for one am not happy to make this assumption but then again I am looking at systems where differences of  2 permil in d13C-values are significant. I might feel different if expected differences were of the order of 15 permil and a deviation of a few permil wouldn't have an impact on conclusions drawn.


Best,

Wolfram



________________________________________
From: Stable Isotope Geochemistry [[log in to unmask]] On Behalf Of Brian N Popp [[log in to unmask]]
Sent: 26 January 2008 18:08
To: [log in to unmask]
Subject: Re: [ISOGEOCHEM] GC/C-IRMS

I worried about formation of copper and nickel fluorides until I spoke with Marilyn Fogel who asked me how much fluoride really ends up going into the reactor tube with each compound? The answer is not a lot even though compound specific nitrogen isotopic analyses of amino acids requires high column loading. We have performed 100's of injections using a single reactor tube. Yes, they do not last as long as a reactor tube used strictly for compound specific carbon isotopic analysis of non-fluoride derivatives, but we found the benefits of TFAA outweigh the shortened reactor life.

We also found the thinkness of the stationary phase improves chromatography. The SGE BPX5 30 m x 0.32 mm id x 1 um gives excellent peak shape and separation for compound specific nitrogen isotopic analyses of amino acids. The chromatography was way better than we got using an HP Ultra2 50 m x 0.32 mm id x 0.5 um even though both are 5% phenyl, 95% methyl siloxane.

Brian

> My advice to you if you choose to accept it is NOT to use
> trifluoroacetylation to derivatize AAs. I know this is one of the
> best derivatization methods for AAs to obtain a sharp, well
> resolved GC chromatograms BUT it is also a sure-fire way to
> exhaust your oxidation reactor very fast and irreversibly (the
> formation of copper and  nickel fluorides makes a re-oxidation
> impossible).