Dear Wolfram,
many thanks for your prompt and detailed answer. You are right by your
argumentation, but the facts are not unkown. I would like to add something.
The MTBSTFA-derivatisation is very quick and an one step method. The
problem is the instability of the samples, which sets in after about just
40 to 48 hours. Two other problems are the possible interaction of the
derivates with the GC-column (we observed a deterioration of the seperation
of the column after 200-300 injections) and the molecule size. This is the
cause of the trouble of the last aa in the chromatogramm- the temperature
of the column is relativly high and you have problems with the stability of
the compounds.
By the TFA you don't have all these described problems. Here is the problem
the irreversible poisoning between the metals in the oxidation furnace and
the F. You must change the oxidation reactor after 100 to 150 injections.
But the price for 2-3 new ox.-furnaces is relativly lower as for a new
GC-column. By the reduction furnace (Cu) we never saw this effect.
Our experience is that the TFA-method is the best suitable derivatisation
for the measurement of Arg. It is a very interesting aa for many problems
in the metabolism research.
It is possible, that we have found special problems by the measurements of
nitrogen. The sample size is much bigger then the size for corbon.
Sometimes it is difficult to disregard the chemical and the physical limit
of the coloumn and to receive enough nitrogen for the isotopical
measurement. At the moment D. Hofmann and myself prepare a paper about
different sample preparation techniques for the nitrogen-determination of
aa by GC/C-IRMS.
By the way I cannot confirm the described effect from CO2 and CO. I think
you didn't have enough oxygen in the furnace or you used an unqualified
temperature.
More details you can find in:
"Methods for investigation of aa and protein metabolism" edited by A. E.
El-Khoury; chapter 7: "The use of GC-C-IRMS for the analysis of stable
isotope enrichment in nitrogenous compounds", C.C. Metges and K.J. Petzke,
1999 by CRC Press
D. Hofmann et al., Isotopes Environm. Health Stud., 31, 367-375, (1995)
D. Hofmann et al., J. Mass Spectrom., 32, 855-863, (1997)
Best regards,.
Matthias
>I'm afraid I beg to differ.
>
>TBDMS may be one of the most convenient, quick and easy-to-use
>methods of derivatisation but is not the best method from an
>analytical point of view, especially not for GC/C-IRMS (and neither
>for GC-MS in my book since the fragmentation channels are dominated
>by sort of McLafferty type re-arrangements of the silyl groups).
>
>(a) All tBDMS does is to make everything very apolar thus
>obliterating chemical differences that could otherwise be exploited
>chromatographically. For example, the tBDMS derivatives of aspartate
>and 4-hydroxyproline show very similar gas chromatographic behaviour
>and can only be resolved employing complex temperature programming
>(and electronic pressure control).
>
>(b) With tBDMS one restricts oneself to the use of apolar stationary
>phases such as SE30 and SE52 / SE54 (100% dimethylpolysiloxane and
>5% diphenyl, dimethyl polysiloxane, respectively).
>
>(c) TBDMS adds an excessive carbon load to one's sample (12 carbons
>for a neutral AA). Example: in leucine for every mol-equivalent of
>N2, 36 mol-equivalents of CO2 are generated. It could be argued that
>this shouldn't influence d15N measurements (i.e. no tracer dilution)
>but alas that is not the case. The excessive carbon load can lead to
>localized, short lived pyrolysis spots in the oxidation furnace thus
>producing CO rather than CO2. CO is not trapped by the obligatory
>cold trap between reduction furnace and IRMS thus entering the ion
>source at the same time as N2 (isobaric interference).
>
>(d) TBMS derivatives of AA are not stable (although the extent of
>their rate of decay seems to be linked to sample history = sample
>origin and sample preparation). I received tBDMS derivatives of AA
>(protein hydrolysate) which despite being stored in the fridge showed
>sample deterioration from week to week, even day to day. However,
>adding some MTBSTFA and heating always brought the peaks back.
>
>As mentioned previously, TFA derivatisation of AA is probably the
>best method from a gas chromatogrpahic point of view. Nice,
>crisp, sharp peaks and you can doe enantioselective GC, too. However,
>you cannot change the law of physics (or chemistry for that matter),
>Captain. TFA will wear down the oxidation catalyst by irreversibly
>poisoning the Pt and by irreversible formation of CuF (red, mp 908 C)
>and NiF2 (sublimates at 1000 C in HF).
>
>I, personally, am not prepared to accept the risk that results might
>be tainted because of this. Especially after I have seen that the
>combustion efficiency suffers almost immediately. Co-injection of Leu
>(as N-TFA-i-propylate) and methyldecanoate (both compounds have the
>same number of carbons) showed a reduced CO2 yield for the former
>compared to that for the latter.
>
>So, it's NAP or ethylchloroformates for me. Both are stable, easy to
>prepare (especially the latter; takes only 10 to 15 minutes) and add
>only 5 carbon per neutral AA. The AAs are still polar (as they
>should) yet volatile and can be chromatographed on medium polar to
>polar stationary phases (OV1701, OV17, OV225, BPX70). The crossbonded
>varieties are stable enough for GC/C-IRMS purposes, and with
>oxygen-free He can be safely used up to 330 C, even 360 C at a push.
>
>
>
>Best regards,
> Wolfram
>
>
>
************************************************************
Matthias Gehre
Centre for Environmental Research Leipzig-Halle
Laboratory of Stable Isotopes
address:
Leipzig Halle
Permoserstr. 15 Theodor-Lieser-Str. 4
D-04318 Leipzig D-06120 Halle/Saale
Germany Germany
Tel.: ++49 (341) 235 2252 Tel.: ++49 (345) 5585 240
Fax.: 2625 Fax.: 559
e-mail: [log in to unmask]
*************************************************************
|
