I am surprised that it took so long for Wolfram to answer or anyone with the correct response.
[U-2H]Palmitate will be resolved via GC or LC from palmitate due to inherent differences
between 2H-palmitate and palmitate on the column. In fact, you only need FID-GC or a regular HPLC
to measure the 2 species. As Wolfram points out, no IRMS technique is useful. This is a job for
either GCMS or LCMS. The sensitivity by these methods is excellent.
If you had [U13C]palmitate, then GC-C-IRMS would provide the good sensitivity because there
would be very little GC separation between the 13C-palmitate and the palmitate, but that is not what
Do you have GCMS or LCMS available?
The tailing is not a problem of the GC as much as it is of the pyrolysis system as you are
introducing a large amount of 2H.
On 5/21/2016 11:33 AM, Wolfram Meier-Augenstein (aps) wrote:
> Dear Isabelle,
> As previous respondents tried to intimate, analysing 2H-uniformly labelled palmitic acid (or methyl palmitate for that matter) by IRMS is less than ideal.
> IRMS was (is) designed to measure stable isotopic composition at near natural abundance level, say < 1 APE. Even if your IRMS instruments permits changing cup resistor values to adjust amplification of the major and minor ion currents according to enrichment, in your case this would not help.
> Even with 1H/2H contribution from the methyl group, the level of 2H enrichment will result in the majority of H2 formed being (2H)2; conversion of 1 unit of U-2H labelled methy palmitate may theoretically yield 15 units of (2H)2 (m/z 4), 1 unit of 1H2H (m/z 3) and 2 units of (1H)2 (m/z 2). A more likely scenario is its conversion will yield 14 units of (2H)2 and 3 units of 1H2H.
> So, the standard set up of measuring m/z 2 and m/z 3 will not work. Adjusting IRMS settings to capture m/z 3 and m/z 4 will not help either since the m/z 4 channel will be not just capture (2H)2+ but He+ too.
> To check on the purity of your U-2H labelled palmitate you are better off using a standard GC/MS system. The mass spectrum should show a molecular ion M+ at m/z 301 and a fragment ion at m/z 270 through loss of the methoxy derivative CH3O.
> For your in-vivo studies I would suggest you analyse your samples (muscle lipid extract) by GC/MS in the first instance. (A) GC/MS is much more sensitive in terms of limit of detection of compound amount (concentration) than GC/IRMS. (B) Even accounting for dilution of U-2H labelled palmitic acid with biogenic palmitic acid and subsequent breakdown and recycling within the body, the level of 2H enrichment in your target compound may still be too high for IRMS.
> -----Original Message-----
> From: Stable Isotope Geochemistry [mailto:[log in to unmask]] On Behalf Of SUBSCRIBE ISOGEOCHEM Isabelle Chery
> Sent: 21 May 2016 15:49
> To: [log in to unmask]
> Subject: [ISOGEOCHEM] D31 Palmitic Acid
> Dear Marylin, Willi, Bill and Brian
> Many thanks for your answers.
> In fact the D labeling is not 100% and with derivatization there are some H added to the mixture. So the mass 3 is significant and is directly proportional to quantity injected (standard curve over 6 concentrations linear at R2 0.99), even if not representing a 100% of the molecule. Moreover the final analysis will be done on human muscle lipid extracts in which the D31 is really at low concentration in a complex lipid matrix.
> The problem Isabelle faces is that the peak she which obtain is not sharp any more and is tailing. It is tailing only for D31 and for non of the other fatty acids of the mixture.
> Few weeks ago the peak and shape were perfect. We have changed injector, cut the column, change the pyrolysis oven, the syringe. We are stuck.
On 5/21/2016 4:02 AM, wbrand wrote:
> Dear Brian,
> as many of you have experienced, memory is a bit difficult to predict from first principles. For
> GC columns, some small amount of many compounds can remain on the column for a longer time,
> contributing to column bleed. When the same chemical is injected again, a compound-specific
> washout effect can replace some of the old material and contribute to the new peak. This is a bit
> like water exchange with the last layer on a metal surface, which cannot be pumped away. However,
> these are rather small effects and should be curable with some special memory treatment (e.g.
> additional injection of some neutralizing mixture). The largest effect that bothers most is the
> apparent tailing right after a highly enriched compound has left the column, prominently visible
> on the ratio trace. The tailing itself is always present (the memory function is not different for
> different isotopologues), but the tailing in this case changes the isotopic composition of the
> baseline. This changing baseline is what causes difficulties for the next couple of eluting
> compounds and renders quantification a bit more difficult. Once the ratio trace is back to
> 'normal' the effect should be gone.
> Regards Willi
> On 5/20/2016 18:28, Brian N. Popp wrote:
>> Willi and others
>> I am curious. How long will it take to get rid of the "blank" created by analysis of a
>> perdeuterated compound before one can return to natural abundance compound specific hydrogen
>> isotopic measurements in this system?
>> On 5/20/2016 5:35 AM, wbrand wrote:
>>> If your D31 palmitic acid is indeed 100% deuterium labeled you would only see mass 4 (D2+) and
>>> some fragment ions at mass 2 (D+). Mass 4 will be a huge peak, as large as the usual mass 2 peak
>>> (H2+) for the non-labeled acid. If you have some hydrogen (protium) left in your palmitic acid,
>>> most of it will show as mass 3 (DH+). I suspect that there is probably quite a lot of this,
>>> which will saturate the mass 3 channel. Moreover, gas chromatography for these H-substituted
>>> palmitic acids will be smeared a bit, which is why I would expect a broader GC peak for mass 3.
>>> You would probably not see this prominently on the 13C peak.
>>> Regards Willi
>>> On 5/20/2016 17:16, Marilyn Fogel wrote:
>>>> Are all of the hydrogens in the molecule deuterium? If so I don't think this is a
>>>> chromatography issue.
>>>> I am not sure how the collector will handle a completely deuterated compound. Perhaps others
>>>> will chime in.
>>>> Marilyn Fogel
>>>> Sent from my iPhone
>>>>> On May 20, 2016, at 7:56 AM, Isabelle Chery <[log in to unmask]> wrote:
>>>>> Hello all,
>>>>> I would like to analyse a uniformly labelled fatty acid with Deuterium (D31 Palmitc Acid)
>>>>> after derivatization to be volatile as methyl ester by Gc pyrolyse IRMS.
>>>>> I use a Zebron ZB Wax capillary GC column to separate different Fames (C14:0 to C22:0). The
>>>>> injector is at 220°C and the high temperature conversion reactor is at 1420°C.
>>>>> The D31-Palmitic acid is totally separated from the natural Palmitic acid by GC.
>>>>> I have beautiful peaks for natural abundance of FAMEs (mass 2 and mass 3) but for the
>>>>> D31-Palmitc acid, the peak shape (only mass 3 is present) is trailing and is not symmetric.
>>>>> I have changed the insert, the syringe, cut the column, and put a new reactor tube. But the
>>>>> problem is always being.
>>>>> Does anyone could help me?
>> Brian N. Popp, Professor
>> University of Hawaii, SOEST, Department of Geology & Geophysics
>> 1680 East-West Road, Honolulu, Hawaii 96822
>> Office - (808) 956-6206; Fax - (808) 956-5521
> Willi A. Brand, Stable Isotope [log in to unmask]
> Max-Planck-Institute for Biogeochemistry (Beutenberg Campus)
> Hans-Knoell-Str. 10, 07745 Jena, Germany Tel: +49-3641-576404
> P.O.Box 100164, 07701 Jena, Germany
Dwight E. Matthews, Ph.D.
Professor of Chemistry and Medicine
The University of Vermont
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