Hi Paul and others,
My key concerns with any calibration are in creating an adequate span, so that one can more accurately identify normalizations with slope and intercept. For isotopic normalizations, I use two different running standards with different deltas, prepared to yield a consistent signal. For elemental, I prepare one standard, generally a pure chemical like acetanilide to yield a range of signals. The elemental calibration standard could be used to imply linearity corrections, but I don't currently do that here. All these standards, plus blind QC samples, are included in a single sequence. When possible, a similar matrix and C:N ratio is selected. My QC samples are always just like the unknowns. If I say I will provide all this data, then I must include standards for each type of calibration. For me, this makes standards often comprise of up to 1/3 of a sequence. But it gives me higher confidence that my data is accurate.
I calibrate all running standards developed or acquired by our laboratory to current international reference materials as defined at http://www.ciaaw.org. The importance of span goes all the way back to this primary calibration. Note the international scale recently changed because of this. So I calibrate with at least three of the international RMs, always including the internationally defined/recommended anchor points. I check calibrations at least annually, and periodically include blind samples of RMs and report their outcome using my running standards.
For every sequence, all reference gasses, standards and samples are prepared to have similar amplitude at the range where my instruments have the least linearity bias. I have found the bias doesn't occur consistently for me. So I try to avoid linearity corrections by only relying on delta values that are within a tested amplitude range.
All my calibrations are then fit with simple least squares, and if needed, an hourly drift correction if applied.
I heavily rely on LIMS, developed by Ty Coplen of the USGS for the bulk of all my data storage, normalization, reporting and billing. It allows one to easily correct data based on one or many standards, using OLS to make the best fit and apply linear drift corrections. If your standards do not allow for an accurate slope estimate, or you want to just use an average offset, you can force the slope of the normalization to 1. I know data isn't always perfect, and post spreadsheet corrections for linearity and multiple drifting standards can still be used with LIMS. I prefer to be a stickler with sample submissions and sequence building so that I avoid complications trying to correct data later.
Don't get me wrong, I think there are some pretty elegant spreadsheet solutions out there. With LIMS I have found for the most part I don't need them. For good data quality, I think we need to realize we can only correct as much as our standards and running conditions will allow.
Cheers,
Ben
Benjamin Harlow
Manager, Stable Isotope Core Laboratory
Washington State University
School of Biological Sciences
G-81 Eastlick
Pullman, WA 99164-4236
Office: 509-335-6161
Lab: 509-335-6154
Fax: 509-335-3184
Laboratory for Biotechnology and Bioanalysis (LBB2) Stable Isotope Core
http://www.isotopes.wsu.edu
-----Original Message-----
From: Stable Isotope Geochemistry [mailto:[log in to unmask]] On Behalf Of Andrew Schauer
Sent: Thursday, October 30, 2008 12:11 PM
To: [log in to unmask]
Subject: Re: [ISOGEOCHEM] CN standards C/N ratio
Paul raises most or all of the important issues related to CN EA work. Its an easy measurement to make and a very difficult one to calibrate properly, as outlined by Paul. I believe the general rule of thumb is, make your standards as similar as possible to your samples (i.e. fish calibrates fish, plant calibrates plant, soil etc etc). With all of the analytical sources of error, hows does one calibrate their in house standards to those reference materials that set the scale? It seems to me that USGS40 and 41 were an attempt to have more appropriate reference materials than NBS19 / LSVEC for d13C and IAEA-N-1 / USGS32 for d15N? Ultimately we all must be on VPDB and Air to report our d13C and d15N, respectively.
andy
Andrew Schauer
ISOLAB
Department of Earth and Space Sciences
University of Washington
Seattle, WA 98195
206.543.6327
[log in to unmask]
----- Original Message ----
From: stableisotopes <[log in to unmask]>
To: [log in to unmask]
Sent: Thursday, October 30, 2008 11:01:24 AM
Subject: [ISOGEOCHEM] CN standards C/N ratio
To ever analyst analyzing 15N and 13C with an elemental analyzer (EA)
and isotope ratio mass spectrometer (IRMS):
I have been following the thread (bottom of this email) on standards for
13C 15N calibration.
For the purposes of my discussion:
1) I refer to the use of two standards with an equal mass of N or C but
different different isotope ratios, for example USGS 40 and USGS 41, as
"scaling" standards, for calibrating the instruments scale.
2) I refer to the same standard at different masses as "linearity"
calibration or checking. This is the change of isotope ratio with size.
3) I refer to the same standard of the same mass analyzed periodically
throughout the analysis as "drift" correction. This is to correct for
drift with time.
I agree completely that we need a set of two standards that have high
and low 15N and 13C delta values so that we can scale the instruments.
The problem is that so far the urea, Acetanilide and glutamic acids have
C/N ratios of 6/1 or less, which is much lower than 90% of the samples
that we analyze. Most of our samples are plant or soil material, with a
C/N ratio of 15 to 30/1. Our experience is that the change of linearity
with size of the combined EA/IRMS system can be significant and must be
corrected for on every analysis. This should not be confused with
linearity checks of the reference gas alone in the IRMS. I have found
several researchers who assume that since their IRMS reference gas was
linear compared to changes in size that different sized samples from the
EA should be as well. When they checked the combined EA/IRMS response
to different sizes of the same standard they discovered that the
response was not linear, and size dependant. This effect seems to
change as the EA and IRMS age, and may change from day to day.
In addition, we have found that the EA/IRMS combination can drift over
time, even with instruments with reference gas injection, so drift
correction standards at regular intervals are required.
Therefore we find it necessary to do analyzes with drift correction
standards every 12 samples, 10 variable weight standards in the analysis
to check for linearity, and additional standards to check scaling. We
usually drift correct and check linearity with standard sets of NIST
1547 peach leaves, which we have found to be uniformly ground out of the
bottle down to 0.2 mg weight, have a C/N ratio of 16, and are available
in over 100g quantities. We then check the scaling with another suitable
quality control standard at a different isotope ratio but similar
material and C/N ratio to the samples we are analyzing. Even this amount
of standards means that we have 30 standards in a typical overnight
analysis of 90 unknowns, resulting in 120 total tins for analysis.
If we were to try to use glutamic acid we would have to weigh out two
sets of standards to get the N and C into the same range as the
samples. This means that we would now have to weigh out 40 drift and
linearity standards for the analysis, plus additional standards for
scaling. This results in almost as many standards as samples in the
samples tray and is completely unrealistic for most work.
I have had several researchers explain that they get around this problem
by; a) analyzing the samples first on a normal EA for %N and %C; b)
weighing out two aliquots of the sample at specific weight for either C
or N analysis so that for an analysis each sample set has very similar
mass of N or C; c) analyzing the samples in two separate analyzes, one
for N and one for C. The problem with this approach, apart from the
fact that it does not lend itself to analysis of samples sent in by
researchers not present at our facility, is that this requires three
analyzes, %, dN and dC, for one sample.
All our experience in ecosystem science work has shown that inevitably
the noise level of the experiment is in the field sampling, and that to
get the noise level of an experiment down one should take and analyze as
many field samples as possible. Therefore it is much more productive to
analyze three field samples each for a combined %, dN and dC than to
analyze one sample three separate times for %, dN and dC.
This raises the question as to how other labs deal with the problems
described above, and if they have found suitable standard material with
isotope ratios of N and C at different ends of the scale but with
suitable C/N ratios.
I am looking forward to input on this.
Paul Brooks
Arndt Schimmelmann wrote:
> Dear Hilmar and those with interest in acetanilides and ureas with 13C
> and/or 15N enrichment,
>
> I thank Ty Coplen for pointing out that USGS 40 and USGS 41 L-glutamic
> acids already provide suitable reference materials with two different
> 13C/12C and 15N/14N ratios for two-point-calibrations. These materials
> should serve the purpose well for most labs. However, I would be happy
> to send test aliquots of Indiana University's acetanilides and ureas
> with 3 levels of 13C and/or 15N abundances to qualified laboratories
> who wish to participate in an informal multi-laboratory comparison.
> Please contact me directly and do not reply to the entire list.
> Thank you for your interest,
> Arndt Schimmelmann
>
>
> Prof. Dr. Hilmar Förstel wrote:
>> Dear Arndt Schimmelmann,
>> Thanks for your comment in a public forum. Does it make sense to make
>> a laboratory comparison? A official round robin test may need too
>> much effort.
>>
>> Yours Hilmar Foerstel
>>
>>
>>> Hello Lora and others,
>>> The use of a single batch of isotopically known acetanilide for d13C
>>> and/or d15N calibration is problematic because single-point
>>> calibration does not account for differences in attenuation of
>>> delta-scales among different mass-spectrometers (i.e. slope). We
>>> should calibrate to the VPDB scale in a way that it correctly
>>> reflects NBS-19 (+1.95) and L-SVEC (-46.6) values. This was
>>> explained by Coplen et al. (2006) New guidelines for d13C
>>> measurements. Analytical Chemistry 78 (7), 2439-2441. The rationale
>>> is the same as for VSMOW and SLAP in hydrogen isotope systematics.
>>> It follows that for internal calibration one should use pairs of
>>> organic reference materials that express significant isotopic
>>> differences. Our lab is currently developing acetanilides with 3
>>> levels of 15N abundance (delta15N ca. +1.2, +19.6, and +40.6 permil;
>>> for standards we use the IAEA- N-1 and IAEA-N-2 ammonium sulfates).
>>> We could not obtain 13C-enriched acetanilide. Instead, we are
>>> developing 3 batches of urea with different levels of 13C and 15N
>>> abundances. A similar project is generating 4 nicotines with
>>> different levels of 13C and 15N abundances for GC-IRMS. These
>>> materials should be ready by the end of this year. Some compounds
>>> are sufficiently characterized already now, but not yet listed on my
>>> website (http://mypage.iu.edu/~aschimme/hc.html). Please contact me
>>> if you have interest.
>>> Best regards,
>>> Arndt Schimmelmann
>>>
>>> Lora L. Wingate wrote:
>>>
>>>> I am using Acetanilide for a C/N weight percentage check standard
>>>> on our new Costech EA coupled to a Delta V Plus. I am interested
>>>> as to whether there is a consensus as to its value for d15N and
>>>> d13C. I have run it alongside and calibrated it relative to IAEA
>>>> and USGS standards, but I am finding that my Acetanilide values are
>>>> different from one lab's data report that I had the opportunity to
>>>> look over. I'd be interested in knowing what values other labs are
>>>> getting. Or, if it is a moot point and Acetanilide isn't deemed a
>>>> reliable isotopic standard, despite the reproducible results we
>>>> obtain. Thanks for any insight, and off list replies are welcome.
>>>>
>>>>
>>>>
>>>> Lora L. Wingate
>>>> University of Michigan
>>>> Department of Geological Sciences
>>>> Stable Isotope Laboratory
>>>> 1100 North University
>>>> 1013 C.C. Little Building
>>>> Ann Arbor, MI 48109-1005
>>> --
>>> Arndt Schimmelmann, Ph.D.
>>> Senior Scientist
>>> Indiana University
>>> Department of Geological Sciences
>>> Biogeochemical Laboratories
>>> 1001 East 10th Street
>>> Bloomington, IN 47405-1405
>>> Ph (812) 855-7645
>>> home (812) 339-3708
>>> FAX (812) 855-7961
>>> e-mail: [log in to unmask]
>>> personal home page:
>>> http://www.indiana.edu/~geosci/people/faculty2.php?n=schimmelmann
>>> home page of Biogeochemical Laboratories:
>>> http://www.indiana.edu/~geosci/research/biogeochem/
>>>
>
>
--
Mr. Paul D. Brooks,
Dept. Integrative Biology MC3140,
3060 Valley Life Sciences Building,
UC Berkeley, 94720-3140.
[log in to unmask]
phone (510)643-1748
FAX (510)643-1749
http://ib.berkeley.edu/groups/biogeochemistry/
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