Wolfram 

Good point, my earlier comments were based strictly on nitrogen isotope analyses of amino acids. As yet, I have no experience with carbon isotopic analyses of individual amino acids.

Brian

----- Original Message -----
From: Wolfram Meier-Augenstein <[log in to unmask]>
Date: Sunday, January 27, 2008 1:02 am
Subject: Re: GC/C-IRMS
To: [log in to unmask]

> 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).
>