Carbon & Nitrogen sample preparation

Carbon and nitrogen samples samples are typically weighed and pelleted by researchers prior to submission to UCSC SIL to be analyzed by CN-EA-iRMS. This is an introduction to CN-EA-iRMS sample preparation for common sample types with instructions for documenting and shipping samples to UCSC SiL for analysis.

Before starting a project, please consult UCSC SiL to determine project feasibility and determine optimal preparation protocols. In person training will be provided for UCSC users.

CN-EA-iRMS sample pre-processing

For proper subsampling and pre-treatment protocols, it is generally advisable to consult methods from peer-reviewed literature relavent to your specific field of research.

Drying in a freeze drier (best) or 50-60 °C oven is an essential pre-treatment for CN-EA-iRMS analysis. Weights for sample amount generally refer to dry weight. A drying oven, freeze drier, and vacuum oven are available for use by UCSC researchers. Please contact UCSC SiL to arrange for the use of these facilities.

Homogenization is a common pre-processing concern for researchers. Note that homogenization does not necessarily improve single measurement precision in CN-EA-iRMS analysis and may be superfluous. Homogenization is typically applied in order to achieve accurate unbiased subsampling of samples significantly larger than the optimal sample amount. For homogenizing plant samples, please refer to notes on grinding plant samples.

Inorganic carbon removal is a common CN-EA-iRMS pre-treatment for soils, sediments, rocks, marine animals, macroalgae, and filtered marine samples. Please refer to notes and procedures for inorganic carbon removal for recommend pre-treatment protocols specific to some of these sample types. Do not remove inorganic carbon from soil, sediment, or rocks with direct application of hydrochloric acid.

Lipid extraction is another common CN-EA-iRMS pretreatment, especially for animal samples. A Dionex Automated Solvent Extractor (ASE) available to UCSC researchers for CN-EA-iRMS sample pre-treatment.

Filter samples are samples collected as a filtrate on a filter in the lab or the field. CN-EA-iRMS analysis of this sample type requires some special consideration. If you are considering a filter project, please be sure to refer to notes on CN-EA-iRMS filter samples before collecting or pelleting filter samples.

CN-EA-iRMS sample amount

In CN-EA-iRMS analysis, a single and pelleted sample produces simultaneous results for C and N amount (μg C, μg N, C:N and if sample weight is known wt%C and wt%N) and stable isotope ratios (δ¹³C and δ¹⁵N or atom%¹³C and atom%¹⁵N).

Optimal sample amount is determined by CN-EA-iRMS analysis type (specifically, whether or not δ¹⁵N is important to the project) and sample type. In general, ~50 μg N gives optimal results for δ¹⁵N when matrix content is insignificant. When δ¹⁵N is not important, smaller sample amounts may give superior results. Note that δ¹⁵N in this discussion refers to measurement of samples with natural ¹⁵N abundance, and not to ¹⁵N-labeled samples which are typically reported in atom%¹⁵N.

The following CN-EA-iRMS optimal sample amounts table differentiates some common sample types based on these three CN-EA-iRMS analysis types.

Note that slightly higher inter-sample accuracy may be achieved by weighing samples to a narrower size range than the size ranges reported in this table. For example, weigh animal tissue samples to 900-1000 μg instead of 600-1100 μg to improve inter-sample accuracy. Or weigh leaf samples to 3000-3300 μg.

Also note that samples significantly smaller than the optimal amounts specified here routinely produce useful data. For example, protein-rich animal tissues like hair or collagen as small as 250 μg routinely yield very good data with only modest loss of precision. And for yet smaller samples, a nanoEA-iRMS system is available for the production of high-precision data. If you are sample limited, please consult UCSC SiL to discuss the best approach for analyzing small samples.

CN-EA-iRMS optimal sample amounts table

Tin capsules and plastic sample trays

Samples prepared for CN-EA-iRMS measurements are weighed and pelleted in tin capsules (also referred to as tin boats). The tin serves two important purposes in CN-EA analysis, first containing the sample in the EA autosampler as a pellet with the proper volume, and then combusting exothermically with a finite amount of oxygen in the EA oxidation reactor to raise the temperature of the sample to ~1500 °C so that organic bonds break spontaneously. Based on these two constraints, the NC2500 EA at UCSC SIL is optimized for samples pelleted in 30-35 mg of tin. At UCSC SiL, these are 9x5 mm cylindrical tin capsules purchased from Costech Analytical (part number 41061 $13 for 100 capsules or part number 41077 $26 for 250 capsules). Comparable products are available from EA Consumables. For some sample types, using a different capsule design may be very helpful. Notably, weighing whisker samples into 12x6x6 mm tin rectangular boats (e.g. EA Consumables SKU D5023) is significantly easier than weighing into cylindrical capsules. If you are using a different tin product to prepare pellets you should ensure that the capsules or boat is not significantly different than 30-35 mg of tin.

Pelleted samples are organized in 96-well trays (called 96-well plates by biologists). Trays need a lid designed to work with the tray. Round-bottom wells are easier to work with than flat-bottom wells. These trays are available from many suppliers. A pack of 10 trays is available for $41 from EA Consumables, SKU E2079. If you have a very small number of samples or if you do not have easy access to 96-well trays, organizing samples into labeled microcentrifuge tubes works well.

Documenting CN-EA-iRMS samples

When you submit samples to UCSC SiL for analysis, please share a spreadsheet file corresponding to the samples that includes information about sample IDs, amounts, and tray locations. Please limit sample IDs to one column and don not use special characters other than dots, dashes, and underscores. This spreadsheet can be shared as a Google Sheet, Excel file, or CSV file.

An image of the basic spreadsheet format is pictured below. You can find a link to this Google Sheet here. Use File > Make a Copy to create your own version of this file.

Keep any physical notes of sample weights for your records. Please do not ship these to UCSC SiL.

UCSC researchers who have already received in person training from UCSC SiL may refer to CN-EA-iRMS run setup workbooks for animal samples and green plant or macroalgae samples.

Weighing and pelleting samples in tin

For precise wt%C and wt%N data, samples should be weighed using a microbalance precise to ±0.002 mg or better. If precise wt%C and wt%N data are unnecessary, then samples may be weighed using a microbalance precise to 0.1 mg. This lower precision in sample weight will not effect the accuracy of either δ¹³C, δ¹⁵N, µg C, µg N, or C:N. UCSC SiL has a microbalance available by reservation for approved users.

Weighing and pelleting samples in tin is available at an extra cost as a service through UCSC SiL. This service is available on a case-by-case basis with prior approval from UCSC SiL and typically adds significant return time for results. The cost of weighing and pelleting service is $2.00 per sample (or $3.08 with overhead when funded through non-UC based grants). The weighing and pelleting service does not include any additional sample pre-treatment or subsampling labor.

For weighing and pelleting <4 mg of sample material UCSC SiL recommends a 'double-crimp & cube' technique. This technique requires some practice to master but almost always produces good results. Samples pelleted with this technique have minimal ripped foil, no jammed autosamplers, and optimal EA combustion.

Technique for weighing and pelleting CN-EA samples <4 mg

For weighing and pelleting samples >4 mg of sample material (soil, sediment, and rock) into 9x5 mm tin capsules UCSC SiL recommends using a 5 mm diameter manual pellet press. This custom piece of equipment is simple to use and produces excellent pellets for up to ~80 mg of sample. A manual pellet press is available for use in UCSC SiL.

If you don't have a 5 mm diameter pellet press, you (or your friendly local machine shop) can make one by drilling a 5 mm diameter hole into an 1/2" height aluminum block with a drill press. To make a piston to fit into the hole for pressing the pellet, turn 1/2" end length of a >5 mm diameter aluminum rod in a lathe so that the diameter is slightly less than 5 mm.

Technique for weighing and pelleting CN-EA samples >4 mg

Packaging and shipping sample trays

In the unusual case that your CN-EA project necessitates preparing samples in capsules smaller than ~33 mg tin (i.e. 3.5x5 mm tin capsules), there is a possibility that pellets will escape their tray wells. There are two solutions to this problem. The most effective, but also more expensive, solution is to purchase silicone seals (EA Consumables SKU EA2044 or EA Consumables SKU EA2079) to cover tray wells. The less expensive solution is to cover the tray wells with several layers of aluminum foil before securing the lid. The foil forms to the shape of the wells, sealing in the pelleted samples. The matte finish side of the foil should be in contact with the samples, as the mirror finish side contains undesirable oil residues and aluminum particulates. After adding foil, tape the lid and package as indicated above for normal ~33 mg tin capsules. The disadvantage of aluminum foil packaging is that an induced static charge can sometimes lift small samples out of their wells when the foil is removed.