Representative samples of plant material are oven-dried at 60 degrees C for 48 h, ground to pass through a 1 mm screen using a Christy Turner Lab Mill*. Triplicate subsamples (2-5 mg) of the ground sample are weighed into tin foil capsules and analyzed for total C and N using a CHN combustion analyzer. Protocols for analyzers used are listed below.

Materials

• Plant grinder (such as Christy Turner Lab Mill*)
  
• 7 mL plastic scintillation vials
  
• Tin capsules
  
• Forceps
  
• Micro spatula, scooped
  
• Sample tray
  
• Electronic microbalance
  
• Standards (acetanilide, atropine or phenacetin**)
  
• Blind standard (plant standard, such as wheat)
  
• CHN analyzer

Procedure

Prepare plant material for analysis:

1. Mix the dried plant sample and break up any large portions into smaller pieces that will fit through the inlet chute.
   
2. Feed the plant material slowly into the inlet chute on the plant grinder.
   
3. Store ground sample in labelled 7 mL plastic scintillation vial.

Prepare sample:

1. Always use forceps to handle tin capsules. Fingers and hands are sources of C and N contamination.
   
2. Place an empty tin capsule on the scale of the microbalance. Close the housing doors. Tare the capsule weight.
   
3. Remove capsule and place on marble block. Using a scooping spatula, pick up 2-5 mg of the ground plant sample and place inside the capsule. Weigh the capsule and sample to ensure it contains an appropriate weight of sample material.
   
4. Return capsule to marble block and use forceps to first roll back the free end of the capsule and then to fold the capsule into a small ball, making sure no sample leaks from the sealed capsule.
   
5. Place folded capsule on the scale, close the doors, and record the sample weight by pressing “print” on the balance instrument panel to link the sample mass via computer software to the selected cell on the data spreadsheet.
   
6. Place capsule in its designated well in the sample tray.
   
7. Always clean the forceps, spatula and marble block with lab wipes between samples.

Prepare analytical sample run:

1. Start run sequence by preparing a “bypass” sample containing material with a high C and N content anticipated for samples (well #A1). Record the well number for each sample in the appropriate spot on the data spreadsheet.
   
2. Next add a “blank” consisting of an empty tin capsule (well #A2).
   
3. Follow with four replicates of a known standard, increasing the weight of sample with each replicate (0.2-0.3mg; 0.5-0.6 mg; 1.0-1.2 mg; and 2.0-2.8 mg). These samples are used to calibrate the analyzer (wells #A3-6).
   
4. Follow with one blind standard (e.g., wheat), prepared as above for plant sample (well #A7).
   
5. Continue the run sequence with samples to be analyzed, preparing three analytical replicates for each sample. Include a blind standard approximately every 12 capsules (wells #A8 onward).
   
6. Store sample tray in desiccator until analyzed. Follow analyzer protocol.

*Prior to 2009, a Wiley Mill and Tecator Impact Pulverizer were used to grind plant material.

** Prior to 1990, cyclohexanone-2,4-dinitrophenylhydrazone was used as standard.

Calculations

None needed. The CHN analyzer reports C and N as the percentage of element in the dried sample (g C or N per 100 g sample). Values reported in the KBS LTER data catalog are the average of the triplicate analytical replicates.

CHN Analyzer Protocols

Carlo Erba NA1500 Series II Combustion Analyzer Protocol
Costech Elemental Combustion System CHNS-O (ECS 4010) Protocol