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.

CN-EA-iRMS analysis types

δ¹⁵N ✓

δ¹⁵N is important

CN-EA-iRMS analysis type:

Natural abundance N isotope ratioswhich also includes results for:Natural abundance C isotope ratiosand C and N amount

Results include:

δ¹⁵N δ¹³Cμg C μg N C:Nwt%C wt%N (if weighed)

δ¹⁵N ✕

δ¹⁵N is not important

CN-EA-iRMS analysis type:

Natural abundance C isotope ratiosand/or C and N amountbut does not include results for:Natural abundance N isotope ratios

Results include:

δ¹³Cμg C μg N C:Nwt%C wt%N (if weighed)

¹³C or ¹⁵N label

Samples are isotopically labeled

CN-EA-iRMS analysis type:

Unnatural abundance ¹³C and/or 15N isotope ratioswhich also includes results for:C and N amount

Results include:

atom%¹³C atom%¹⁵Nμg C μg N C:Nwt%C wt%N (if weighed)

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

Animal tissue (protein-rich)

Protein rich animal tissues include muscle, skin, collagen, blood, serum, liver, hair, whisker, fur, feather, scales, deep sea proteinaceous coral, and many invertebrates.

600-1100 μg

δ¹⁵N ✓

600-1100 μg

δ¹⁵N ✕

250-1000 μg

¹³C or ¹⁵N label

Animal tissue (lipid-rich or sugar-rich)

N abundance of fatty or sugary samples may be very low.

Consult SiL

δ¹⁵N ✓

600-1100 μg

δ¹⁵N ✕

250-1000 μg

¹³C or ¹⁵N label

Dentine

1300-1500 μg

δ¹⁵N ✓

800-1100 μg

δ¹⁵N ✕

250-1000 μg

¹³C or ¹⁵N label

Green plant tissues or macroalgae

Green plant tissues include leaves, stems, shoots, fine roots, and root nodules.
If plant or macroalgae samples are significantly decayed or significantly charred, please consult UCSC SiL to determine optimal sample amount.

2700-3300 μg

δ¹⁵N ✓

600-1100 μg

δ¹⁵N ✕

250-1000 μg

¹³C or ¹⁵N label

Woody plant tissue

N abundance in wood is too low for accurate δ15N analysis at UCSC SIL.

N/A

δ¹⁵N ✓

600-1100 μg

δ¹⁵N ✕

250-1000 μg

¹³C or ¹⁵N label

Fruits or seeds

Consult SIL

δ¹⁵N ✓

600-1100 μg

δ¹⁵N ✕

250-1000 μg

¹³C or ¹⁵N label

Soil or Sediment or Rock

Optimal sample amount depends on relative organic and matrix content. If C or N abundance are known, consult UCSC SiL to determine optimal sample amount. For high-matrix samples, amounts up to ~80 mg may be measured.

40-50 mg

δ¹⁵N ✓

10-12 mg

δ¹⁵N ✕

N/A

¹³C or ¹⁵N label

Filter (25 mm diameter)

Please see Notes on CN-EA-iRMS filter samples for a discussion of subsampling filters.

½ or 1

δ¹⁵N ✓

½ or 1

δ¹⁵N ✕

½ or 1

¹³C or ¹⁵N label

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

1. Use a pair of curved fine tip forceps, a pair of straight fine tip forceps, a Hayman-style microspatula, and an anvil (a clean hard working surface). Wash your hands. Gloves are not recommended for non-hazardous substances.

2. Tare one tin capsule. Capsule weights vary, so capsules must be tared individually.
Please use tin capsules that weigh 30-35 mg and only one capsule per sample unless otherwise specified by UCSC SIL.

3. Scoop sample material with the tip of the triangle end of the microspatula.
When subsampling fine grained material from a vial, look through the side of the vial to use only the tip of the microspatula.

4. Rotate the microspatula with sample to vertical over the open capsule. Tap the other end of the microspatula with forceps or another instrument to release all of the sample into the capsule.

5. Adjust the amount of sample as necessary, then record the final weight of the sample.
You do not need to record the weight of the tin capsule.

6. Hold the straight forceps in your dominant hand, with the curved forceps in your other hand. Close the straight forceps on the top edge of the capsule, closing the top of the capsule.

7. Keeping the straight forceps closed, place the capsule on the anvil with the bottom of the capsule towards you. Roll the top of the capsule towards you so that the capsule is folded over twice.

8. Use the flat edge of the closed curved forceps to hold the sample in place (think of this as a 'foot'), then pull the straight forceps out of the double-fold.

9. Press down on top of the double-fold to form a double-crimp. This should form a rectangle on top of the capsule.
Great job! Your weighed sample is now sealed into the capsule.

10. To pelletize the capsule, use the rectangle of the double crimp as a template. Use your forceps to carefully and gently fold the bottom of the capsule containing your sample on top of the rectangle. Take some care with this step.

11. Rotate the rectangle 90° on the anvil. Fold the tin up and around your sample by holding the center of the rectangle down with the flat edge of the closed curved forceps, then pulling the long edges of the rectangle on top with the straight forceps.

12. To form the pellet into a cube, grasp the sides of the pellet with the straight forceps. Press down on top with the flat edge of the closed curved forceps. Rotate 90° and repeat.

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

1. Use one pair of forceps, a Hayman-style microspatula, a 5 mm diameter pressing block with piston, and an anvil (a clean hard working surface). Wash your hands. Gloves are not recommended for non-hazardous substances.

2. Tare one 9x5 mm tin capsule on a microbalance. Insert the tin capsule into the hole in the pressing block so that it is sticking out ~5 mm on the top (about ½ to ⅓ of the capsule should be sticking out of the top). Tare each new capsule individually.

3. Using forceps, open the top of the capsule sticking out of the block. The top can be opened all the way to flat, or opened into a cone shape, which can help funnel sample material into the capsule.

4. Scoop sample material with the tip of the triangle end of the microspatula. When subsampling fine grained material from a vial, look through the side of the vial to use only the tip of the microspatula.

5. Rotate the microspatula with sample to vertical over the open capsule. Tap the other end of the microspatula with forceps or another instrument to release all of the sample into the capsule.

6. Remove the capsule with sample from the block and return to the microbalance to check the sample weight. Adjust the sample amount as necessary and then record the final sample weight.

7. Return the capsule into the hole in the pressing block. Using forceps, fold opposite sides of the capsule over the open hole in the capsule.

8. Rotate the pressing block 90°, then repeat folding opposite sides of the capsule over the hole.

9. By making four folds, you should form a square on the top of the capsule. The corners of the square may extend slightly past the circumference of the hole in the pressing block. This is normal.

10. Hold the piston in your dominant hand, and very firmly hold the pressing block down on the anvil with your other hand. Form a pellet by pressing straight down hard with the piston. Do not rotate the piston or press up and down more than once.

11. Lift the pressing block up and press gently with the piston to eject the pellet.

12. An ideal pellet will have smooth walled sides and a concave top. Move the pellet into the sample tray by holding one of the points on the top rim of the pellet with forceps.

Packaging and shipping sample trays

Tape and label

To prepare 96-well trays for shipment, secure the lid to the top of each tray with four short pieces of tape attached perpendicular to the edge of the tray. This will effectively secure samples.
Please do not use excess tape on the trays.

Stack and wrap

If you have more than one tray, stack the trays. Wrap the tray or trays as you would a gift using a layer of bubble wrap, foam padding, or other padding material and tape tightly.
Once wrapped, a single tray or short stack of trays may be easily and safely shipped in a typical mailing envelope or small box.

DO NOT

Do not use any Parafilm to package trays. This product is made of plasticizing pthalate compounds that can alter the isotopic composition of your samples.
Do not add notecards or aluminum foil inside of trays unless otherwise specified.

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.

Please ship samples to this address:

UCSC - Earth and Planetary SciencesStable Isotope Laboratory1156 High Street, E&MS C512Santa Cruz, CA 95064ATTN: Colin Carney cpcarney@ucsc.edu

Carbon & Nitrogen sample preparation

CN-EA-iRMS sample pre-processing

CN-EA-iRMS sample amount

CN-EA-iRMS analysis types

δ15N ✓

δ15N ✕

13C or 15N label

CN-EA-iRMS optimal sample amounts table

Tin capsules and plastic sample trays

Documenting CN-EA-iRMS samples

Weighing and pelleting samples in tin

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

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

Packaging and shipping sample trays

Tape and label

Stack and wrap

DO NOT

Please ship samples to this address:

δ¹⁵N ✓

δ¹⁵N ✕

¹³C or ¹⁵N label