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Standard Operating Procedures

Water Fluorination and Preparation for IRMS Analysis

Introduction

Lorax water 17O preparation describes how to run the prep line used to fluorinate water to form O2 which is subsequently run on Lorax, a MAT 253, for oxygen isotopes. Use the Quick Start section if you already have a good idea of what you are doing. See below the Quick Start section for more thorough descriptions.

The line needs to be baked out prior to each run and prior to a long dormant period. It will take you about 30 minutes to start up, 2 hours to condition it line, and then about 1 hour per sample. You will need to top off liquid nitrogen in all dewars during a narrow window prior to each sample.

Safety

This method generates hydrofluoric acid (HF) and users must know where the HF is at all times. Appropriate precautions should be taken to protect yourself against the cold and hot temperatures. Wear safety glasses and leather gloves when handling liquid nitrogen.

Quick Start

  1. bake out the line and change the septum
  2. allow traps to cool
  3. prepare water-sample vials
  4. start filling in daily log
  5. start labview
  6. freeze T1 when labview indicates its time
  7. prepare syringe and autosampler
  8. enter run information into labview
  9. continue with the daily log
  10. keep running until all cold-fingers are full
  11. add liquid nitrogen to dewars at appropriate time throughout the day
  12. when run is completely finished, drop all dewars
  13. allow traps to completely dry, they can NOT be wet when you start the bake out
  14. click the start button to stop the run, then click manual
  15. if the Lorax is done, remove octopus / squid to take to mass spec
  16. install octopus / squid that was just removed from mass spec
  17. the line should be left such that T2 is being pumped on

Exhaustive Protocol

Baking out the line

The 17O water line should be baked out prior to each run even if it was baked out and then left dormant. Baking out the line involves heating up Trap 1 (T1), Trap 2 (T2), and the octopus / squid. You should also consider changing out the septum early in the bake out process.

Getting the bake-out started - Start by placing one of the cold-finger manifolds (octopus or squid) into position. If they are both busy, place a glass stub in the open ultra-torr port. Then place the metal box around T1 supporting it with a lab jack. Adjust the two-piece lid on top of the box. Then wrap T2 in foil. Plug in the heat tape that is coiled around the space between V3 and T1. Now turn on the buttons labeled 'T1 - Bake' and 'T2 - Bake'. They will turn red when they are on. Turn the 'Manual' button on (it will turn green). Make sure V0 and V2 are open (green). VA should be 'right'. VB should be 'left'. V1 should be closed (dark green). V3 and V4 should be open. Really, the only two valves you should have to bother with are V3 and V4. If you did install the octopus or squid AND you don't have samples inside those cold-fingers, attached the air pressure lines from the cold-finger manifold to the prepline ports making sure cold-finger 1 is attached to port 1 and so on until all 8 are attached. Place the cold-finger heater collar around the tips of the manifold and support it with the other lab jack. Turn the dial that the heater collar controller to high. Now open all eight cold-finger valves using labview buttons S1-S8.

Changing the septum - With a pair of tweezers, press in / down at the center of the ultra-torr injection port. Slowly loosen the ultra-torr fitting until you hear a pop (that’s the helium). Remove the cone shaped needle guide and red septum. Obtain a new septum from the drawer below the prepline. If you place the new septum in the port it will just pop out. Instead, thread the tweezer tips through the hole of the ultra-torr fitting, place the new septum over the injection port, set the needle guide on top of the septum and hold it all in place down with the tweezer tips, and finally slide the ultra-torr fitting down the tweezers while maintaining downward pressure on the needle guide and screw the ultra-torr nut into place. Tighten the fitting, making sure the septum doesn’t get folded or caught in the threads.

When is the bake-out done? - Scroll down on the labview screen until you see G2 and G1 time series figures. These should both show an increasing trend followed by a decreasing trend with time as gases are released from the surfaces that are being heated. The bake-out is complete when G1 and G2 are near background levels (G1 == 5e-5 Torr, G2 == 4 mTorr). If they are close to these values but still slightly above and you are in a hurry, it is probably ok to turn the heaters off and move forward with your day.

Shutting down the bake-out - Once you are satisfied with the G1 and G2 readings, turn off the buttons labeled 'T1 - Bake' and 'T2 - Bake' (they will turn from red to gray). Turn off the cold-finger heater collar but rotating the dial to off. Unplug the heat tape that is wrapped up between V3 and T1. With heat-protective gloves (located under muffle furnace), remove the T1 metal box, the foil wrapped around T2, and the collar around the cold fingers. BE CAREFUL, these are 500 °C! Allow these all to cool to less than 100 °C.

Preparing your water samples

The line allows you to fill 8 cold-fingers in one run. Ideally one of these is a standard carefully chosen for its similarity to your samples. Prior to injecting a sample, the line injects two column conditioners and one full conditioner. This is, then, a maximum of 11 total injections in one run. The autosampler allows you to inject n number of times per vial where the n is identical among all vials. Work out how many injections you want per vial before loading waters into vials. One strategy is to load one vial for every single injection (11 vials) and to have the first 3 vials be the standard you are going to use and the next 8 vials are samples plus one standard. Another strategy is to load 6 vials total, the first two vials are the standard, the other 4 are samples. This will condition the line with the standard and have the first cold finger be that standard. You can also do one vial if you are running only a single water for the entire day.

After you decide on a strategy, load 200 µL of sample / standard into vials that have 300-µL fused inserts. Choose your standard based on waters that have a known Δ17O which can be viewed here. Ideally, the standard you choose also is close in δ18O.

Place the vials in order into the autosampler tray from 1 to n.

Prepare the autosampler

The autosampler is old and prone to misalignment. This section describes how to ameliorate misalignment issues and how to set up the autosampler according to how you loaded your waters.

Misalignment seems to be preventable by forcing the autosampler to go through a corrective startup sequence. Start by removing the syringe and setting it aside. On the autosampler control pad, press CLR twice, then INJ twice. The autosampler is now primed to go through an injection sequence. Click the 'Cycle Autosampler' button and the autosampler will beep and begin to move. Turn the autosampler switch that is located on the back of the autosampler main body to the OFF position. Manually move the arm of the autosampler slightly. Turn the autosampler switch back ON and allow it to complete its startup routine. Press INJ twice, click 'Cycle Autosampler' again and watch to see that the arm lines up well with the first vial and the injection port. If not, repeat this paragraph.

You should have already decided how your sequence will run when you prepared your water vials for the day. Adjust autosampler accordingly. Press CLR twice. Set FST to first autosampler position (usually 001). Set LST to last autosampler position (either 7 or 11 in the two examples above). LST will rarely if ever exceed 11. Set CNT to the number of injections per vial (either 2 or 1 in the two examples above). Press INJ twice when you are done adjusting the settings.

Prepare the syringe

The syringe will become dirty and sticky with use. You can choose to clean it as describe below or replace it with a new one.

Remove the syringe from the autosampler by rotating the black syringe-body lock to a vertical position. Find the syringe cleaning kit (it is most likely in 302A near one of the Picarro lasers). Fill the 'helper' syringe that is in the kit (not the one you just removed) with the 1% HCl solution and set aside. Place the syringe you just removed from the autosampler into the HCl solution and pull the plunger up half way or as far as you can. Use the 'helper' syringe and apply HCl to the top of the autosampler syringe (e.g. where the plunger meets the syringe glass body). Set the helper syringe aside. Continue moving the autosampler syringe up and down while it is in the HCl solution until plunger can be removed from syringe body (this may take a while and you must be patient and gentle). Wipe off plunger with a wipe. Reinsert the plunger into the body and continue moving the plunger up and down while still in the HCl. Remove the plunger again and wipe it off. Inject all HCl back into HCl container. Fill the autosampler syringe with DI water by pulling plunger all the way to the top. Release this water into a wipe, and repeat ~5 times. Rinse heavily with the same DI while keeping syringe tip in the DI water. Reinsert the, now clean, syringe into the autosampler and indicate complete on autosampler controller. Rinse the 'helper' syringe with DI water in the same way. If you can’t remove the plunger during cleaning or break it or it was already broken, obtain a new syringe from drawer under the prepline. Discard the old syringe to the broken glass box in 303B.

The Daily Log

The daily log allows you to enter the starting state of the prepline as well as injection information so we can predict when the cobalt fluoride column is about to run out.

Start by entering the date (yymmdd - year year month month day day) then your name. Enter the helium cylinder pressure as well as the pressure leaving the regulator. Do not run if the helium cylinder pressure is below 100 psi. Enter the cobalt fluoride column temperature from the LabView screen. Now enter the dates when the cobalt fluoride column was new, when the syringe was new, and when the septum was changed. You can also skip a few columns and enter manifold (octopus or squid) as well as something that describes the waters you are running (e.g. sample set id). You can also enter the number of vials, injection size, and injections per vial. Copy the formulas for total injected water and cumulative injected water. If the cumulative injected water is nearing or exceeding 600 µL, it is probably nearing time for a new cobalt fluoride column.

The columns you skipped over need to be filled out at certain stages of the run. After the bake-out is complete and the traps are cooled, enter the pressure readings from both G1 and G2. When the script is collecting O2 from the Full Conditioner, make note of and enter the Ball Valve Flow in mm. The mL / min column is a formula conversion.

The LabView script

Automation of the prepline is controlled by LabView. The main part of the script screen shows a diagram of the prepline as well as buttons that help you process your samples. The lower right area is where you will enter run information or where run information is presented to you. You can scroll down below the main screen to see figures showing running time series of vacuum gauges and thermocouples. Below is a description of how to interact with the script and what exactly it will be doing.

The name of the script that we are currently using is 'Water_17O_PrepLine.vi'. If you must reboot the computer or LabView appears to be stopped, you must restart this script. You can open 'National Instruments LabVIEW' to start the main program. You will be prompted to choose a script to open. Choose 'Water_17O_PrepLine'. The script is not yet running. To start the script, click the play button (white sideways triangle) located at the upper left of the screen.

When the script is running you can start a run or manipulate the line manually. If you intend to manipulate the line manually, make sure the start button is off and the manual button is on. You should have the manual button on when doing a bake-out.

Get a run going

To start a run, it is assumed that you have baked out the line, loaded your waters, prepared the autosampler, prepared the syringe, and started filling out the daily log. Make sure the manual button is off and click start. Read through the Status window to ensure you are ready to start. Click “Pre run complete” V2 will close. Place the large blue dewar in place under T1. Fill the T1 dewar with liquid nitrogen. The T1 dewar should raise all the way up to the horizontal Ni tube. When T1 is completely frozen (agressive boiling has stopped), click “Freeze T1”. A new checklist will appear.

Enter your sample IDs. You only need to enter the sample name itself, however, the formal sample ID format is YYMMDD_n_yourID, where YYMMDD is todays date, n is the cold finger number (1-8), and yourID is something descriptive. If yourID is a standard water (e.g. SW), then simply enter SW. It is critical that you don't name yourID of your samples with a name already taken by our standard waters. Again, the only text you enter in Sample ID is the name of the water. LabView will prepend this name with YYMMDD_n_. You will type the full formal SampleID into the mass spec software later.

Continue filling out the lower right side of the screen. 'Last sample' should be the number of samples (not counting conditioners). If you want to end your day early, choose something lower than 8. 'User Comments' will clear after each sample and you can use this field to make comments about individual samples.

Next, place the medium sized dewar T2 and fill it such that the liquid nitrogen is BELOW the bottom of the trap. Put the manifold trap in place under the cold fingers and fill it to the top before raising it completely with the lab jack.

You are now ready to click 'start samples'.

Throughout the run, top off the dewars when V1 and V2 are closed. This will be a ~2 minute window, during step 9 (about 42 minutes after the injection). You can also top off dewars anytime, by very slowly adding LN2 keeping He pressure within +/- 5 of 90 mm

What is the LabView script doing?

Here are the exhaustive details of what the script is doing after the 'start samples' button is pressed. In this section parenthetical values indicate how long that step takes.

Immediately after 'start samples' is pressed, the autosampler is triggered to perform one column conditioning injection (90 seconds), a second column conditioning injection is performed (90 seconds). The injected water is allowed to move through the column and converted O2 vents to the hood (1800 seconds, 30 minutes). Total time 33 minutes .

Now the main loop is entered and processing of samples begins starting with a Full Conditioner. A full conditioner is identical to a sample except it is not saved into a cold finger. The main loop starts by closing V2, switching VA and VB and raising the T2 dewar and then allowing 2 minutes for the trap to cool and for you to fill the dewars (2 minutes). VA and VB are positioned left and right, respectively such that helium is now filling T2. V1 opens, the autosampler is triggered and an injection begins. The script then allows for the conversion to O2 and collection on the molecular sieve trap T2 (30 minutes). V1 closes, VA switches back to right and V2 opens to set the helium flow back to default. V3 and V4 open while VB toggles left and right to begin evacuating helium from T2. VB toggles left and right to partially evacuate the space between VA and VB (the entire helium evacuation routine takes 3 minutes). V3 and V4 close and the lifter lowers to begin warming T2 for a yield measurement on G1 (3 minutes). The appropriate cold finger valve opens and transfer of O2 happens (5 minutes). The cold finger valve closes, anything that did not freeze into the cold finger is pumped away, and T2 is allowed to cool from transfer temperature to room temperature (2 minutes). Total time for each sample 45 minutes.

After all injections are complete

Drop dewars and let traps thaw and heat tape dry. This will take a while, but can be helped with compressed air. Turn off “Start”. Turn on “manual”. Close black manual valve on manifold. Undo each of the 8 green air hoses. Do this on the manifold side of the valve. Keep manifold upright! Transfer it to the mass spec. Take the manifold that was connected to the mass spec, and attach it back to the prep line. Attach green air hoses for both mass spec and prep line manifolds. Turn on heater for both manifolds and pump out the neck. Follow mass spec instructions for mass spec manifold. Once traps are thawed and heat tape is dry, proceed to bake-out procedure above.

Troubleshooting

Low or no yield - Low or not O2 produced could be caused by clogged syringe, super leaky septum, spent cobalt fluoride. If you notice a low or no yield, make note of the ball meter flow rate. If you do not have flow, check for a leaky septum or perhaps you have a clog in the line. If you do have flow at the ball meter, remove the syringe and see if you are getting water in and out of it. If you are not getting any water out of the syringe, replace it. If everything so far checks out, perhaps the cobalt fluoride is spent in which case you need a fresh cobalt fluoride column.

Syringe broken - If the autosampler becomes misaligned and smashes the syringe needle, go through the prepare autosampler section above. Then replace the syringe with a new one. The dead syringe goes in the broken glass.