Cryogenic Extraction of Water from Stems, Leaves, or Soils

Protocol

AUTHOR

Louis Santiago: modified from T.E. Dawson, J.R. Ehleringer, M.F. Allen, and T.J. Jones

OVERVIEW

This is the standard method used to extract water from leaves, soils, and stems for later analysis of either hydrogen or oxygen isotope ratios.

MATERIALS/EQUIPMENT

To construct the vacuum line

    • Vacuum pump (Rotary Vane vacuum pump from Edwards or others)
    • Glass tubes (can be purchased or blown)
    • Swagelok Ultra Torr Fittings (for attaching sections)

Other

  • Leaf, soil, or stem samples (kept frozen until analysis)
  • Pyrex collection tube
  • 1 inch ignition (sample) tube
  • Beakers 500 mL + of water
  • Liquid nitrogen LN2
  • Dewars (vacuum flasks)
  • Glass wool
  • Analytical balance
  • Record notebook
  • Lab tape for labels
  • Post-it notes
  • Kimwipes
  • Sample storage vials with lids e.g. Vacutainers
  • Deionized water
  • Parafilm
  • Freezer or refrigerator for water storage
  • Drying oven at 65∘C

UNITS, TERMS, DEFINITIONS

Cryogenic

mTorr

Liquid Nitrogen – LN2

Deionised water – DI

Parafilm

PROCEDURE

Note: Directions for sample extraction should be repeated for each individual line.

  1. Leaf, soil, and stem samples should be kept frozen until analysis.
  2. NOTE: The night before the sample is run, make sure the vacuum pump is turned on.
  3. Attach a Pyrex collection tube and a 1 inch ignition (sample) tube to the cryogenic vacuum extraction line. Swagelok connectors (Swagelok Ultra Torr fittings) should be screwed finger-tight. Grease all o-rings. Replace any o-rings that are cracked, broken or flattened. Make sure Swagelok connections are clean and dry.
  4. Let the extraction line pump down until it holds a low and steady vacuum. The main line should have a vacuum between 0-30 mTorr.
  5. Fill beakers to around 400 mL of water. Place on hot plate and turn on. Start low and warm up to around medium (4 or 5).
  6. Fill dewars with LN2. Use LN2 to fill flasks.
  7. Just before extraction, bring sample vials to room temperature. Wipe off the condensation on the vials and weigh the vials for a pre-extraction mass (includes jar and lid, add glass wool if soil to keep soil particles from moving through the vacuum line). Record pre-mass.
  8. Give your sample a number in place of it’s label and record the label and number in the notebook. Write the number on the sample vial, the sample lid, and a piece of lab tape which will be stuck to the line gauge.
  9. Check each line independently and make sure each gauge stops between 0-50 mTorr without leaks. This is the “zero-point of the day”. Write it on a post-it and put it on the line for later reference. Zero the gauges if necessary. Record initial vacuum level. Close the lines and check back after 5 minutes. Record closed vacuum. If there is a large leak, re-check all seals and o-rings. Do not use until there are small to no leaks (0-30 mTorr per minute is acceptable, less is better).
  10. Un-cap your vial, wipe the lid with a piece of Kimwipe (put the Kimwipe into the vial with the sample). Place the sample (in vial but without lid) into the ignition tube and freeze with liquid nitrogen (LN2). Continue to freeze the sample in LN2 until the sample is clearly frozen (about 10-15 min).
  11. Once the sample is frozen, open the vacuum valve and evacuate the glass extraction manifold. Let evacuate for around 10 min. After evacuation, close the vacuum valve, isolating the sample from the vacuum line. Record freezing vacuum.
  12. Remove the LN2 from the ignition tube. Replace it with a 400 mL beaker containing deionized water. Immerse the ignition tube as far up as possible (sample should be completely below water level). Hot plate should be around 4 or 5 (medium level hot). Turn on heating tape (to level between 5 & 6).
  13. Turn hot plate to HIGH. Keep the water level constant during the entire extraction process.
  14. Once the water is at a boil, freeze the sample collection tube with LN2. Begin with only the very tip of the collection tube immersed in the LN2. Slowly move LN2 flask up the collection vial. If you run out of room you may need to melt the ice down and refreeze. Make sure to refreeze slowly so as not to break the tube.
  15. Boil for 60-90 minutes. Recommended extraction time for soils is 40-45 min., stems is 60-70 min. and leaves around 60 min. If moisture condenses on the vacuum line walls, use a hair dryer or heat gun (keep heat gun on low <2) to evaporate it and drive it over to and then to the bottom of the sample collection tube.
  16. You can usually tell when all of the moisture has been extracted from the sample, because there will be no further moisture condensation on the vacuum line walls. One way to check this is to briefly (for 5 seconds) immerse the collection tube farther up with LN2. If bubbles form on the inside of the tube then condensation is still occurring so keep extracting. Once condensation has stopped the sample is finished. Turn off the hot plate and remove the boiling water. It is better to leave the sample on “too long” than “too short.” So may want to leave sample on line between 15 min. to 1 hour after condensation is gone.
  17. Remove the LN2 from the sample collection tube and remove the finished tube from the extraction line. Then apply a label, parafilm the top and let it thaw. Turn off heating tape.
  18. Remove the sample. Put the lid on and weigh the sample (including jar, lid, and glass wool) for post-mass.
  19. Weigh the water, sample storage vial, and parafilm. Decant the thawed sample into a storage vial, parafilm the top and store in the freezer or refrigerator. Weigh the storage sample with the water and parafilm.
  20. Use the pre-mass, post-mass, and mass of the water (collection vial mass with water – collection vial mass – parafilm mass) to determine efficiency.
  21. Place finished samples without lids into the drying oven at 65° for 48 hours. When finished weigh the sample. Use this to double check efficiency.

The sample is now ready for hydrogen isotope and/or oxygen isotope analyses. See Isotope methods.

NOTES AND TROUBLESHOOTING TIPS

Headings for a lab notebook:

Sample # Sample ID Line # Type Collection Vial (g) Sample Pre-Mass (g) Sample Post-Mass (g) Sample Dry Mass (g) Collection Vial + Water (g) Parafilm (g) Water (g) Vacuum Initial (mTorr) Vacuum Closed (mTorr) Vacuum Freezing (mTorr) Date

Recommended extraction time for

  • soils is 40-45 min.,
  • stems is 60-70 min. and
  • leaves around 60 min.

Dewars (vacuum flasks)

Swagelok Ultra Torr fittings

Heating tape

Vacutainers

LITERATURE REFERENCES

Ehleringer, J.R. & Dawson, T.E. (1992) Water uptake by plants: perspectives from stable isotope composition. Plant, Cell and Environment, 15, 1073-1082.

West, A.G., Patrickson, S.J. & Ehleringer, J.R. (2006) Water extraction times for plant and soil materials used in stable isotope analysis. Rapid Communications in Mass Spectrometry, 20, 1317-1321.

HEALTH, SAFETY & HAZARDOUS WASTE DISPOSAL CONSIDERATIONS

  • Take extreme care when handling LN2 and ensure you wear suitable gloves

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