Solid Oxygen Isotope Analysis

[DRAFT] Introduction This document outlines how to run Lorax for oxygen isotope analysis of solid materials using the TCEA coupled to our Finnigan MAT 253 (Lorax) via a Conflo III, is set up as a continuous flow isotope ratio mass spectrometer for the analysis and oxygen isotopes. Use the Terse Procedure section if you already have a good idea of what you are doing. Use the Exhaustive Protocol section for more thorough descriptions. Any direction based instructions are only relevant when the Lorax is in its current position (center of room with loading area near fume hood). Safety Appropriate precautions should be taken to protect yourself against the high temperatures of the elemental analyzer and the reagents used therein. Users are expected to know the contents of the SDS for each of the ingredients of the columns and know what to do in case of exposure or spillage. Terse Procedure 1. Prepare and Weigh Samples and Standards – A typical tray of 49 drops should proceed cndtnr_GA1, qtycal_GA1 2x target, qtycal_GA1 1/2x target, empty silver, BaSO4 (standard), Cocoa (standard), GA1 (standard), 15 samples, Cocoa (standard), BaSO4 (standard), and a GA1 (standard). There must be a ref_GA1 between every drop starting after the conditioner. Consult the microbalance method. 2. Previous Run Finished – Check info box at bottom of screen to confirm sequence has terminated and finished. 3. Note Backgrounds – Check current backgrounds, especially CO (m/z 28), N2 (m/z 28), Ar (m/z 40), and H2O (m/z 18). 4. Check Column – Make sure there are no issues with the column. The column should go through only 4 runs before being replaced. If possible, note which run is being done (1-4), to know both when the column needs to be replaced and to note any differences compared to previous runs. 5. Load Samples – Turn on helium dilution, close isolation valve, vent and vacuum autosampler, open autosampler lid, check carousel position, manual advance once, load samples, close and secure lid, begin evacuating autosampler. 6. Complete Sequence Table / Enter Data - Open appropriate sequence. Enter masses and sample / standard identifiers. Ensure your desired method is selected. 7. Purging Autosampler / Carousel – When you are finished evacuating the autosampler, close vacuum, open helium purge barrel valve, vent helium 3 times, open isolation valve. 8. Daily Log – Turn off helium dilution, purge until backgrounds are similar to previously noted, fill out one row in Lorax daily log. 9. Start Run – Close helium barrel valve, highlight rows containing samples and standards if carousel is not full. Click the Start button. Folder Name should be your sample set ID. Leave File Name alone. Export Format should be .csv and File Name should be sample set ID. Click OK. 10. Data/Results - Move ONLY the .csv file from C:\Thermo\Isodat NT\Global\User\Conflo II Interface\Results\* to S:\data\projects\loraxCO\rawdata_new . * is appropriate directory for your run and your project. Open python and type ‘py loraxCO.py’. Create a folder called ‘reduced’ at the same level as ‘raw’. Type comments, look at figures, etc. A reduced data file was placed in the ‘reduced’ folder. Exhaustive Protocol Weighing Samples and Standards Samples and standards are weighed into silver or tin capsules. Consult the microbalance method. The target weight for Lorax is dependent on the material being run. To determine the target weights for your sample material: multiply the atomic mass of oxygen by the number of oxygen atoms and then divide by molecular mass multiplied by 100 to find to find oxygen percentage (e.g. BaSO4: ). Divide 50 by the oxygen percentage divided by 100, and then divide all by 1000. (e.g. BaSO4: ). Divide 100 by the oxygen percentage divided by 100, and then divide all by 1000. (e.g. BaSO4: ). Samples are typically weighed into silver capsules, but we do have tin capsules available. Silver capsules must be kept under vacuum once opened. We have various sizes of capsules depending on the quantity of material you intend to weigh. Its best to weigh samples and standards into the same sized capsule. A typical tray of 49 drops should proceed cndtnr_GA1, qtycal_GA1 2x target, qtycal_GA1 1/2x target, empty silver, BaSO4 (standard), Cocoa (standard), GA1 (standard), 15 samples, Cocoa (standard), BaSO4 (standard), and a GA1 (standard). There must be a ref_GA1 between every drop (standards and samples) starting after the conditioner, (e.g. Cocoa (standard), ref_GA1, BaSO4 (standard), ref_GA1, sample, ref_GA1, sample, etc.). Each drop should have a peak for N and O. Previous Run Check the Information box at the base of the IsoDat Acquisition screen to see if the previous run has finished. The information box is a play-by-play log of events that updates while the instrument is running. If the instrument was previously running and is finished, you should see a few rows indicating that the sequence finished, the last of which will have a green dot with white check mark, a time stamp, the word 'Sequencer', and the text "Sequence Finished !". It is a good idea to view the samples of the previous run as a first cut at checking for proper instrument functionality.  Instrument Preparation Current Instrument State – Before opening the instrument up to change the insert, change columns, or load samples, make a mental note of the CO (m/z 28), N2 (m/z 28), Ar (m/z 40), and H2O (m/z 18) backgrounds and the carrier flow rate with the dilution OFF. Within Isodat Acquisition, change the gas configuration by using the drop-down menu in the lower left corner of the screen. The gas configuration should be CO when running Lorax. Make certain all reference gases are off by clicking any Reference gas dark gray bars in the Conflo window on the left side of the screen. The N2 background should be around 100 mV. Check with past daily logs to compare pressures, backgrounds, and information in MS window in Isodat Acquisition. Replacing the Column – The Lorax column needs to be changed every four runs. If possible, note which run is being done (1-4), to know both when the column needs to be replaced and to note any differences compared to previous runs. Check if the column has any issues, such as the fan being misplaced or not running, the column being out of place, backgrounds being unusually low or high, or needing to replace the column. The following directions are how to remove and rebuild the column… Loading Samples Preparing the Carousel – Click on the Helium Dilution bar in Conflo II III Interface to turn it on. Close isolation valve (arrow pointing towards you), then vent the autosampler by pointing the three-way valve on the lid towards the vent (towards the window). Vacuum the carousel (arrow towards whiteboard) until able to turn silver bolts holding carousel closed. Turn all three slightly then remove fully and open the carousel hatch. Check the carousel for lint and debris, wipe the o ring with fingers to clean and press into place. isolation valve three-way valve silver bolts carousel hatch Autosampler Alignment – Whether you remove the carousel for cleaning or not, you should check the alignment to ensure proper dropping of samples. Ideally, the carousel hole is lined up exactly with the base plate such that you can not see the base plate. The Lorax has a 50-hole carousel, therefore the 50th hole should be aligned with the column and have a visible hole. Before loading any samples, arrange the carousel to have the 49th hole over the column and manually advance once by pressing the "MANUAL ADVANCE" on the costech control box. Costech control box If the carousel is not properly and exactly aligned, such as having any part of the base plate visible for the 50th hole, you will need to make fine scale adjustments with the "jog" switch on the back of the costech control box. Toggling the Jog switch up (Jog +) will very slowly move the carousel in the positive direction (same direction as the manual advance button), while toggling the jog switch down (Jog -) will very slowly move the carousel backwards. YOU MUST ALWAYS FINISH YOUR JOG ADJUSTMENTS IN THE POSITIVE DIRECTION. If you move the jog switch at all, always finish these fine scale adjustments with one last "MANUAL ADVANCE" to ensure proper alignment. This may take multiple attempts to arrange correctly. Loading Samples – Before loading any samples, confirm that all samples from the previous run have been dropped or removed from the autosampler. Using tweezers of your choice, carefully place samples and standards sequentially in the autosampler starting to the right of the hole (which may be position 50; e.g. start loading at position 1). Continue until all samples and standards are loaded. Now is a good time to make final adjustments on your sample capsules if they are very full. If they are touching multiple sides of the autosampler hole, you may want to squeeze them to be thinner and taller. Closing the Lid – Close the hatch and secure the bolts into place, making them finger tight. Rotate the 3-way lid valve to the vacuum position (towards the whiteboard) to pump out the atmospheric air from the autosampler. As it is pumping, you will need to keep tightening the lid bolts, again, to finger tightness. IT IS NOT NECESSARY TO USE A TOOL TO MAKE THE NUTS TIGHTER THAN FINGER TIGHT. Evacuate autosampler for 5 minutes. Proceed to the next step while waiting. Purging Autosampler After about 5 minutes of evacuation, close the three-way valve (pointing away from you). Open the helium purge barrel valve. Purge helium by slowly turning the three-way valve (towards window) and then closing it after the hissing of helium stops. Wait until the Purge dial on the side panel is stable before repeating. Repeat three-ish times. End with the three-way valve closed (away from you). Open the isolation valve SLOWLY (arrow pointed up). Purge at least once more before proceeding to the next step. Sequence Table On the left side of the screen, find the Sequence tab under the Browser window bar to open the appropriate sequence. Currently we are using Conflo_CO_v1.seq. Method – The far-right column should depict the method. The first row should be Conflo_CO_v1.met, whereas every row below that should be Conflo_CO_v2_drop_is_ref.met. Amount – For row 1: this is the mass of your cndtrn_GA1. For every row after, this is the mass of your standard or sample. Identifier 1 – This is the name of your standard or sample (e.g. BaSO4, qtycal_GA1, known sample container (e.g. A1, C6, F3)). Identifier 2 –This is your ref_GA1. It should be written as GA1 for all rows EXCEPT row 1, which is left blank. Comment – This is the mass of your ref_GA1 for every row EXCEPT row 1, which is left blank. Daily Log The lab uses a daily log for each instrument or preparation line to allow users a first glance at the readiness of the instrument. By comparing the current state of the instrument to historical states, you are more informed about the instrument and whether or not it is functioning properly and ready to run your samples. Each daily log is web based and browser accessible. No link is provided here by design. Open the browser on the controlling computer and you should see at least two tabs already open. One tab is this SOP and the other is the daily log. If the browser has more than two tabs open, it may have additional SOPs. Use the bookmark toolbar as needed if tabs have been closed. Before Entering Information – Turn off the Helium Dilution, otherwise your information will be incorrect. Purge the autosampler a few more times until the backgrounds are similar to previously noted, from both before opening the carousel and from past daily logs. Wait a few minutes after purging to enter data as it takes time for the machine to retrieve the data. Work through each cell of the daily log. If you are uncertain where to find certain information, hover over the column header tip, denoted by a ⓘ symbol. Make certain to press the 'save to log' button when you are finished entering data. You are welcome to make notes if you have observed something with or done something to the instrument and would like to document that information. Use the "insert note" link at the top of the daily log to make a note. You may enter notes at any time. Start Before starting, close the helium barrel valve. Then highlight rows containing samples and standards if carousel is not full. Click the Start button. Folder Name should be your sample set ID. Leave File Name alone. Export Format should be .csv and File Name should be sample set ID. Click OK. Watch the first few drops of your run to make sure it is running as expected. Data Move ONLY the .csv file from C:\Thermo\Isodat NT\Global\User\Conflo II Interface\Results\* to S:\data\projects\loraxCO\rawdata_new . * is appropriate directory for your run and your project. Open python and type ‘py loraxCO.py’. Create a folder called ‘reduced’ at the same level as ‘raw’. Type comments, look at figures, etc. A reduced data file was placed in the ‘reduced’ folder. Troubleshooting High backgrounds – Use the mini gas leak detector to check for leaks. A helium leak will show as red LEDs. If the autosampler lid is leaking, slowly open the vent valve, open the lid, wipe the o-ring and lid clean, close the lid, close the vent valve. You can also change to Ar (mass 40) and then use the Argon cylinder to spray connections and watch for increases in signal. Try to assess if the high backgrounds are due to an atmospheric leak or are due to something internal. High N2 and Ar might indicate an atmospheric leak. High H2O may indicate a wet GC column and may need baking out. Unusually low backgrounds – If you are getting values twice as low or more in Nitrogen and Argon from the historical values seen in the daily log, there may be a leak large enough to prevent helium from passing into the mass spec. For example, Mass 40 has a 20-30 mV signal when it usually is ~100 mV. However, the most parsimonious explanation for this observation is the three-way valve on the shrek valve switching bank is pointed towards flow meter, rather than mass spec. Computer Crash – If the IsoDat software and / or computer crash, quit everything and reboot the computer. Open Acquisition and turn the source on by clicking the red sun icon in the upper left portion of the window. Suggested Reading TC/EA Operating Manual

Signature

I have read and understood the present SOP and, before starting work outlined in the present SOP, I will complete all required training.