Protocol for clumped isotopes - weighing and running measurements

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Introduction

The following protocol describes how to weigh samples and run clumped isotopes measurements.


Safety Information

Wear cryo-gloves, long sleeve shirts, protection glasses and closed shoes when handling liquid nitrogen in open dewars. Liquid nitrogen is extremely cold. The vapor of liquid nitrogen can rapidly freeze skin tissue and eye fluid, resulting in cold burns, frostbite, and permanent eye damage even by brief exposure.


In the event of spillage of liquid nitrogen on a person: If exposed to liquid or cold gas, restore tissue to normal body temperature, 37°C, followed by protection of the injured tissue from further damage and infection. Remove or loosen clothing that may constrict blood circulation to the frozen area. Rapid warming of the affected part is best achieved by using water at 42°C. Water should under no circumstances be over 44°C, nor should the frozen part be rubbed either before or after rewarming. If in doubt contact Legevakt (phone 116 117 or 113 for emergencies).


If someone gets liquid nitrogen it in their eyes, call 113, wash the eyes with appropriate equipment immediately.


Sample identification and measurement sequence

A run list should first be made, using one of the worksheets in the weighing room (sheets labeled for mass spectrometer). Standards and samples in appropriate numbers and at appropriate positions should be filled in (some may already be listed). Project and project number for billing must be written when applicable.


Weighing in standards/sample material (usually the day before starting a run)

Equipment:

  • Spatula
  • tweezers
  • tray
  • brush
  • weighing boat
  • rack for sample vials, and
  • clean Kiel vials.
  1. Write yourself on the calendar to book a balance in advance. Otherwise, check if the microscale is available.
  2. Determine what mass range you need to use (dependent on run type).
  3. Assemble necessary tools: picking tray, ‘mini-spoon’, brush, tweezers, weighing papers, weighing boat (small aluminium ones most appropriate), racks and appropriate vials.
  4. Clean clumped isotope vials are found in the blue wire racks; NEVER use vials from somewhere else and never mix up clumped isotope vials with any others!
  5. There must be two clean and empty vials at the beginning of the sample rack (pump vials used as place holders and for safety during a measurement run).
  6. Clean the working bench and all weighing equipment with Kim Wipes/brush such that your working area is free from dust and the implements you will use to weigh are clean and free from contaminants.
  7. Do not place vials, racks or other equipment on the anti-vibration table or on top of the microscale to prevent destabilizing the balance.
  8. Be very gentle with the microbalances, they are very sensitive. Do not put undue pressure on the microbalance weighing tray. You WILL break it if you push on it.
  9. Refer to your run sheet for standards and masses.
  10. Find clumped isotope standards ISOA, ISOB, CHALK and RIEDEL in the standard desiccator.
  11. Open the standard bottle only when you take out standard and close it immediately after use. NEVER PUT MATERIAL BACK INTO THE STANDARD BOTTLE AND NEVER POUR STANDARD OUT!
  12. Place the appropriate mass of standard powder or sample onto your weighing boat or paper.
  13. Carefully lead the boat/paper towards the microscale by using a tweezer.
    • If the weight is too large: remove from the microscale and pick/pour the extra grains out on the tray or another weighing paper. You can, and should, reuse standard material in the next aliquot if the tray/paper is clean.
  14. If desired, clean the vial before inserting the sample using clean air.
  15. Turn the vial horizontally then insert the weighing boat/paper as far as possible towards the base of the vial. Turn vertical then pour and carefully knocking the boat towards the wall to transfer all material to the vial.
  16. Weigh the empty boat/paper again to ensure complete transfer.
  17. Tap the vial with a tweezer to make sure all sample material is located at the bottom of the vial.
  18. Repeat procedure from step 10 for all samples/standards.
  19. IMPORTANT! When changing to a new standard/sample use a brush and Kim Wipes to clean all apparatus thoroughly to avoid contamination.
  20. Place the standards back in the desiccator as soon as you are finished.
  21. If you leave the weighing room for a break, make sure to cover all vials to prevent dust and other impurities to fall into the vials. It is good practice to keep all vials covered as much as possible.
  22. Once you are done, clean after yourself and put everything away.


Starting a clumped isotope measurement run

In: Isodat Acquisition

  1. Heat Trap
    • Open up Kiel valve 2 and 71 (hold ctrl)
    • Close Kiel valve 72 (hold ctrl)
    • Set Trap 2 to 140 oC
  2. (YETI) Bake Porapak at 150 oC for 1 hour
    • On temperature controller, to the rear of the Kiel: Hold P long --> OPEr
    • Press P again --> SP
    • Press P again --> SP1
    • Press P again (current set point value will be shown) and use arrows to change temperature
    • Once on correct temperature, press P again and wait until it stops blinking and displays the temperature it is measuring; set 1 hour timer after it has reached 150 °C
  3. (NESSIE) Bake Porapak at 120 °C for 1.5 hours (Nessie)
    • On small temperature control window, press heat (to 120 °C) on software and set timer for 1.5 hrs
  4. Remove small LN dewar to allow to defrost
  5. Removing and re-loading carousel
    • Right click on Kiel window and select ‘Take magazine’
    • Wait for vials to be released and movement of magazine/pistons to stop; select ‘OK’ on pop-up window stating ‘Ready to take magazine’
    • Remove magazine from Kiel and place in holder on counter top
    • Check all samples have been reacted, that acid levels are consistent throughout run and between lines, and that there is no acid discolouration (if these things are noted, let someone know)
    • Place used vials in plastic beaker labelled ‘Climb’
    • Clean drop counters and acid needles using kimwipes, clean o-rings using finger tips (can ‘grease’ o-rings using finger or face oil if desired, but do not use any other oils or greases)
    • Place new vials into magazine and reload into Kiel
    • Right click on Kiel window and select ‘Load magazine’
  6. Post baking: commence cooling following the same steps. On Yeti cool to -40 °C and run can start once Porapak has reached -20 °C. Check that the Porapak does reach -40 °C during initial part of run, well before first sample is transferred through porapak. On Nessie cool to -20 °C and after 30 minutes the run can be started.

In: Instrument Control

  1. Background Peak Scans
    • Open valve 33 (change over valve 2) and valve 25 to allow gas to source
    • Select Peak Shape in third drop down menu
    • Open Options
      • Pre delay = 10 s
      • Start = 9.4
      • Stop = 9.6
      • Step = 0.0002
    • Adjust pressure on bellow until you reach 25 V on mass 44 (+/- 300 mV), wait for one minute after changing bellow position to ensure pressure has stabilised before starting scan
    • Save scan (scan has finished when buttons become available on left hand side)
    • Repeat steps d and e for 20 V (as close as possible, max +/- 200 mV), 15 V (+/- 250 mV), 10 V (+/- 250 mV), and 5 V (+/- 250 mV); wait minimum 30 seconds after moving bellow for all these intensities (pressure stabilizes faster when decreasing)
    • Record the pressure in the below at 20 V on mass 44 in the log book
    • Load background files into Easotope (in Screens -> Data Input, Scans tab; select correct mass spec and select ‘Add Scan’); click ‘Load previous background’, and show regression on 47. Record the pressure on 20 V on mass 47 in Maintenance log spreadsheet and log book
  2. Close valve 33 (change over valve 2) and valve 25 to close off gas from source
  3. Close Instrument Control

In: Isodat Acquisition

  1. Right click on mass 45, select ‘Jump to mass’, change to mass 18
    • Peak Center scan
    • Record V on mass 18 in log book (may have to read off graph if intensity too low to complete Peak Center)
  2. Return to correct mass/focus settings by reselecting CO2-clumped (YETI) or CO2 (NESSIE) on the drop down menu at the bottom of the Acquisition window
    • If you wish to use the 1012 Ω resistor (m47) then select CO2-clumped-low (YETI) or CO2_low (NESSIE)
  3. In the log book record the values for:
    • FV
    • Box
    • Trap
    • Vac (MS)

In: Isodat Workspace

  1. Load previous sequence (or the most recent run you know is similar to yours in terms of run type and sample sequence)
  2. Click ‘Save As’ and change file name to next run number
  3. Type up sequence, including changing run number in sequence
  4. Check location of ref refills and adjust as necessary
  5. Check that correct method has been selected (for example, Kiel LIDI)
  6. Save

In: Isodat Acquisition

  1. Expand bellow to 100 and check it is properly calibrated (by looking at the physical position of the pin inside the track behind the reference gas bellow)
  2. Replace LN dewar (remember to first empty water from the ‘new’ dewar and then transfer the excess LN from the used one).
  3. Load Sequence, highlight rows you want to run and press Start
    • Remember to update the run number (x3)

NB: Runs should only be started after the Porapak has cooled sufficiently (-20 °C on Yeti and after 30 minutes of cooling on Nessie)

In case of lack of gas to perform scans: how to refill bellow

  1. Open change over valve 34 (close valve 33) and close valve 25 to close the bellow off from the source
  2. Expand bellow to 100
  3. Open valves 23, 39, and 22
  4. After 3-4 s close valve 23 (valve 39 should close on its own after a set time)
  5. Open valve 24 and wait until pressure in bellow at 100 = ~35-36
  6. Close valves 24 and 22
  7. Now safe to open bellow to source by opening change over valve 33 (closing valve 34) and opening valve 25. There should now be sufficient gas in the bellow for the background scans.
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