Drying,
grinding, and weighing
(a few do's and don'ts...)
1. Samples must be
dried, ground, and weighed prior to isotope analysis. We do not accept "wet" samples in our lab, so all samples must be dried to constant weight prior to submission. On the very rare occasion, we will accept samples that require drying, but there is an additional cost associated with this, and approval by our lab must be given prior to submission.
2. Large samples must be sub-sampled prior to submission. Isotope analysis only requires milligram quantities of material, so we do not encourge sending entire plants or whole fish (unless very small) for prep work here in our lab.
3. All soils should be sieved to a consistent particle
size (e.g., 40 mesh) prior to grinding and weighing. Soils suspected of containing carbonates may exhibit erroneous
(organic) δ 13C values
unless acid-treatment is performed prior to analysis. We do not automatically acid-treat soils, unless requested.
Drying
of plants, animals, and soils can be accomplished via a drying
oven at 50-60°C for 24-48 h, or via a freeze drier. Grinding
can be achieved via a mortar and pestle, a ball-mill grinder,
a mixer mill, etc. The goal is to improve sample homogeneity
(i.e., isotopic homogeneity) by way of pulverizing the sample
into a fine powder or flour with a consistent particle size.
After drying and grinding, samples need to be weighed into small
tin capsules prior to isotope analysis. Capsule size will
depend on sample weight. For example, 4x6-mm tin capsules
are preferred when sample weights are less than 7 mg; 5x9-mm
tin capsules are used for sample weights between 7 mg and 40
mg; and 9x10-mm tin capsules are used for samples greater than
40 mg. Tin capsules can be purchased from COSTECH
Analytical Inc. (800-524-7219), CE
Elantech (888-232-4676), Elemental
Microanalysis (978-526-8517), Elementar Americas (856-787-0022), or in Canada from Isomass
Scientific (800-363-7823), or from any other company supplying
products and services for isotope-ratio and/or elemental analysis.
Using
a micro-analytical balance, the mass of a sample should be determined
to 3 decimal places on a milligram. For example, you should be
able to record the mass as "2.104 mg". [Please note: if
you are unable to acquire a balance that measures to 3 decimal
places on a milligram, but instead can only use a balance that
measures to 2 decimal places (e.g., 2.10 mg) or to 1 decimal
place (e.g., 2.1 mg), it is important to realize that some precision
may be lost when measuring %C and %N (but not C/N, or δ13C
andδ15N). This
is because %C and %N are calculated using the recorded weight
of the sample. Isotope measurements are not affected by small
weighing inaccuracies because their determination is not weight-dependent...
beyond the assurance that a large enough sample has been combusted
in the EA. Thus if one can live with slightly lower precision
on %C and %N (e.g., ± 2.0%
for carbon as opposed to ± 0.5%)
then acquiring a balance that records to 3 decimal places on
a milligram is not essential.]
Prior
to weighing out the sample, tare the balance to exclude the initial
mass of the tin capsule. Once the appropriate amount of sample
has been placed in the tin capsule, the capsule needs to be "crushed" into
a small ball or square (see figure below - you want the final
sample to look like #4). This can be accomplished by gently applying
force using a pair of forceps, or by rolling the sample gently
between your thumb and index finger.

At no point in time should
you touch the sample or the tin capsule with your bare hands. Powder-less
latex gloves should be used if you plan on "crushing" the capsule
between your fingers. I usually use 2 pairs of forceps to handle
the sample, so that I never have to touch it with my bare hands. Please
note that if a capsule containing a sample falls on the floor,
it should be thrown away. Also, please be sure that material
will not leak from the capsule after it has been crushed, as the
loss of material can affect both the isotope and elemental data
(through incorrect mass determination and subsequent sample-to-sample
contamination). Once the tin capsule has been crushed, please re-weigh
the sample to confirm and record the final mass.
Mass requirements
The required dry mass for simultaneous analysis of δ15Nand
δ13C
depends mostly on the sample type, but also on the nitrogen content
of each sample. Since nitrogen occurs in lower concentrations than
carbon (for most organic materials), the nitrogen content becomes the
limiting factor in dual-isotope measurements. Plants (i.e., 2-5% nitrogen)
and soils/sediments (0.1-1.0% nitrogen) have much lower nitrogen concentrations
than animals (i.e., 8-12% nitrogen), so more material is needed per sample
for plants and soils than for animals. The preference in our
isotope lab is to weigh out 60 micrograms of nitrogen per sample (regardless
of the sample type). This amount of material usually results
in a nitrogen peak of sufficient size for accurately measuring stable isotopes,
but it does not result in a peak so large that it will exhaust the
chemicals too quickly. Since there is usually much more carbon than
nitrogen in organic materials, there should always be enough carbon per sample
for δ13Canalysis
(so long as samples have been properly prepared for δ15Nanalysis).
[Please note: there is both a LOWER limit and an UPPER limit
to the amount of sample that should be submitted in each tin cup. Please
follow the guidelines below for different sample types, and if you
have any questions, do not hesitate to contact the lab
manager for more information.]
To achieve 60
micrograms of nitrogen per sample, one needs to know the approximate
nitrogen content of the material being analyzed. As mentioned above,
plants usually contain less nitrogen than animals, so to obtain 60 micrograms
of N for plants with a nitrogen content of approximately 2%, one would
need to weigh out approximately 3.000 milligrams of dried plant material.
For animals (e.g., fish, birds, mammals, invertebrates, etc.) with a
nitrogen content of approximately 10%, one would need to weigh out approximately
0.600 milligrams of dried material to obtain 60 micrograms of N. For
ease of measurement, we usually ask clients to weigh out plant material
between 4.000 and 6.000 mg. For animal material, we ask that you
weigh out samples between 0.600 mg and 1.200 mg. Below,
there is a small table outlining required sample masses, given the approximate
% nitrogen content of different sample materials:
Milligrams of dried
material to be weighed per sample |
%N of sample |
Micrograms of N needed
per sample for isotope analysis |
Possible
sample types |
60.000 |
0.1% |
60 |
soil |
12.000 |
0.5% |
60 |
soil |
4.000 to 6.000 |
1.5% |
60 |
decaying plant
litter |
3.000 to 4.000 |
3.0% |
60 |
fresh leaves,
algae |
2.000 to 3.000 |
5.0% |
60 |
legumes |
0.600 to 1.200 |
10.0% |
60 |
all animals |
0.300 |
20.0% |
60 |
ammonium salts |
0.150 |
40.0% |
60 |
urea |
If you are unsure of the nitrogen content of your sample (e.g., bacteria,
soils, sediments, FPOM, etc.), it would be best to send a preliminary sample
of the material (in a vial) to our lab so that we could analyze it for you
and recommend an appropriate mass. Another alternative is for you to send
ALL of your samples to our lab to be weighed here, at which point we would
be able to run repeats for you on larger/smaller samples if necessary.
Once weighed, crushed, and re-weighed, samples should be placed into a
96-well cell plate (with cover) for easy storage and shipping. The
following two images show samples that are poorly packed into cell plates
and those that are well packed.
These are poorly packed samples. Note the
scattered bits of organic material all over the tray...
Here's another bad tray. Note how dirty some of the wells are, and how
one sample is broken into two pieces.
Here are some very nicely packed samples. Very compact. No tears or leaks...
We use 96-well polystyrene plates. Cell plates can be ordered (in bulk)
from companies such as Fisher-Scientific
Inc or VWR International. For
smaller quantities, plates can be purchased from COSTECH
Analytical Inc.
Each well in the cell plate has an alpha-numeric position (rows A through
H, columns 1 through 12). Thus, the first well is A1, the next A2, and
so on.
CAUTION! Once you are ready to ship a tray of weighed samples, please check that the space between the lid and the tray is not large enough to allow a sample to jump out of position. This is very important. To test this you could place a dummy sample or two in wells in the middle of the tray, replace the lid, and then shake the tray. If the samples jump out of position, you need to use a different type of tray, or simply press a sheet of parafilm over the samples, prior to replacing the lid. You can also use silicon compression mats for 96-well plates (e.g., VWR# 10011-002).
Cost of simultaneous δ13C and δ15N analyses via EA-CFIRMS
Updated sample costs are listed here
For this price, you get δ15N, δ13C, %C, %N, and C/N for each sample.
There are additional costs for other tasks required in our lab prior to isotope analysis (e.g., sieving
soils, acid-treating soils to
remove inorganic carbon, lipid extraction, solvent washing, grinding samples to a fine powder, weighing samples,
etc.).
For additional prices on other types of analyses conducted at CPSIL, click here.
Turn-around Time
Please contact us for our current turnaround times.
Once the samples have been analyzed, we will send you a copy of the data
via email. A hardcopy will follow by regular mail (if requested) along with the invoice
for services. Included in the data file will be the results of quality
assurance and quality control (QA/QC). For carbon and nitrogen analysis, we use four IAEA standards (IAEA-N1,
N2, CH6, and CH7) to correct (i.e., normalize) the raw isotope data. We
then use a series of elemental standards and various NIST standards to
correct the %C and the %N data. These standards also serve as secondary
checks on isotope data from day to day, and are logged into a datafile
which serves as a long-term QA/QC report. Standards are interspersed throughout
each daily run, to ensure data integrity over the entire analysis. As you
will note on the sample-submission sheet, we ask that you weigh out duplicates
of some samples as a check on sample homogeneity and reproducibility. If
for some unknown reason (e.g., power failure, exhaustion of chemicals,
computer error) a sample is lost during analysis, we will request that
you re-submit the material. Any repeats that requested by our lab will
be analyzed free-of-charge.
Shipping
To send samples to our lab, please click here for
our shipping address.
Additional Information
We run samples for δ15N, δ13C,
%C, %N, and C/N, in continuous-flow mode using a Thermo-Finnigan Deltaplus Advantage
gas isotope-ratio mass spectrometer interfaced with a Costech Analytical
ECS4010 elemental analyzer. We also have a Carlo Erba 2100 elemental
analyzer which can be run in tandem on our Deltaplus XL mass spectrometer.
Helium flow rate is usually set at 110-130 ml/min. Oxygen flow rate
is at 80 ml/min. A standard 3-meter GC column is used (set at 55°C) for
peak separation, in combination with one quartz (combustion) tube filled
with chromium oxide and silvered cobaltous/cobaltic oxide (set at 1020°C)
and one quartz (reduction) tube filled with reduced copper (set at
650°C). Dual
analysis of δ15N and δ13C
usually takes about 7-8 minutes per sample. The average daily run contains
approximately 120 samples (incl. standards). Data are normalized using
4 internationally-accepted isotope reference standards (IAEA CH6, CH7, N1, and N2). Our
main working standard is peach leaves (NIST 1547). External precision
on these standards is ± 0.10‰ or better
for δ13C and ± 0.20‰ or
better for δ15N. δ13C
and δ15N data are expressed
relative to VPDB for carbon, and to AIR for nitrogen.
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