Stable isotope sample submission instructions 

1. Samples must be dried, ground, and weighed prior to isotope analysis. We do not accept “wet” samples. All samples must be dried to constant weight prior to submission. On very rare occasion, we will accept samples that require drying; there is an additional cost, and approval by our lab must be given prior to submission.
2. Large samples must be subsampled prior to submission. Isotope analysis only requires milligram quantities of material, so we do not encourage sending entire plants or whole fish (unless very small) for prep work.
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) ç¹³C 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, 4×6-mm tin capsules are preferred when sample weights are less than 7 mg, 5×9-mm tin capsules are used for sample weights between 7 mg and 40 mg, and 9×10-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, Inc, (888-232-4676), Elemental Microanalysis (855-695-1312), Elementar Americas, Inc. (856-787-0022), or in Canada from Isomass Scientific, Inc. (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.”

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 and δ¹⁵N). 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. Powderless latex gloves should be used if you plan on “crushing” the capsule between your fingers. I usually use two pairs of forceps to handle the sample, so 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.

The required dry mass for simultaneous analysis of δ¹⁵N and 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 δ¹³ Canalysis (so long as samples have been properly prepared for δ¹⁵ Nanalysis).

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 please 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. The table below outlines the required sample masses, given the approximate % nitrogen content of different sample materials:

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.

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.

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 hard copy 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 data file 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 resubmit the material. Any repeats requested by our lab will be analyzed free of charge.

We run samples for δ¹⁵N, δ¹³C, %C, %N, and C/N, in continuous-flow mode using our Thermo DELTA V Plus gas isotope-ratio mass spectrometer interfaced with a Costech Analytical ECS 4010 Elemental Analyzer via a Thermo Conflo-IV. We also run CN samples using our Thermo DELTA V Advantage gas isotope-ratio mass spectrometer interfaced with a Carlo Erba 2100 Elemental Analyzer via Conflo-III. ISP Helium flow rate is usually set at 110–130 ml/min. Oxygen flow rate is 80 ml/min. A standard 3-meter GC column is used (set at between 45 & 55°C) for peak separation, in combination with one quartz (combustion) tube filled with chromium oxide and silvered cobaltous/cobaltic oxide (set at 1000°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.  Data on δ¹³C and δ¹⁵N are expressed relative to VPDB for carbon and to AIR for nitrogen.

We analyze water samples for δ¹⁸O and δD via Cavity Ring-Down Spectroscopy using our Picarro L2140-i coupled to the Picarro fully integrated Autosampler (A0325) and Vaporizer (A0211) for simultaneous D/H and ¹⁸O /¹⁶O ratios measurements of H₂O. 2.0 ml of sample/standard is filtered through 0.2 μm Acrodisc® GHP membrane HPLC certified filters attached to BD® 1ml syringes, and injected into Waters® 12×32 mm screw-top 2 ml vials capped with pre-slit PTFE/Silicone septa. 6 (2 μL) injections per sample/standard are delivered through the liquid autosampler using a 10 μL SGE® microliter syringe (Model 002982) to be evaluated by the Picarro L2140-i. The first 3 injections are rejected and not used in any calculations to minimize any carryover from the previous sample. The means of the last 3 injections per sample/standard are used in the actual calculations. 6 injections of Nanopure water are used in the beginning of the run to check for variations in water ppm measured by the L2140-i. All of our in-house water standards are calculated using VSMOW2 (Vienna Standard Mean Ocean Water 2) (0‰), SLAP2 (Standard Light Antarctic Precipitation 2) (δD -427.5‰ and δ¹⁸O -55.5‰) and GRESP (Greenland Summit Precipitation) (δD -258.0‰ and δ¹⁸O -33.4‰). Normalization and drift calculations are applied to the data set based on known standards added to each run. Check standards (not used in the calculations) are also added to the run to ‘check’ the accuracy of the applied equations. The Picarro L2140-i reported δ¹⁸O precision is <0.025‰ per sample, and 24-hour drift is <0.2‰. The reported δD precision is <0.1‰ per sample, and 24-hour drift is <0.8‰.