Using the Mass Specs--general instructions 1 2 3 4earth.yale.edu/sites/default/files/files/ESCSIS/ESCSIS-man_prelim.pdf · HV= 3.04 (Not 0.3--click on “lightning” icon to turn

Edited on ?/ ESCSIS-man_newest.doc 2/15/12

Earth Systems Center for Stable Isotopic Studies, a Yale Institute for Biospheric Studies center Dr. Peter Raymond, ESCSIS Director; Dr. Glendon Hunsinger, ESCSIS Manager

Using the Mass Specs--general instructions: Please note: You need to be trained on the mass spectrometer and peripheral before running them. You should also examine the mass spec’s and peripheral’s user guides, appropriate application reports from Thermo Finnigan, P. A. de Groot’s “Handbook of Stable Isotope Analytical Techniques” that we have, the “Isogeochem” list server, and, of course, the literature for your area of study. This is only a guide, and is still a work in progress. If you got something to work well, please write down what you did so you can remember it--‘cause I’ll forget it. 1. File locations: Your data and sequence files are now located under:

Finnigan\stuff\<data, sequence, etc.>\<P.I.name>\<your-name>\ With your methods, results workshop, data export templates, etc. under the PI name only: Finnigan\stuff\<method, workshop, etc.>\<P.I.name>\

2. File paths in Isodat are set to data, method, etc., for appropriate heading and the upper most level will show the P.I. names. GC methods are a special issue, see section for GCC. 3. Template folders and files for methods and sequences are present. Please don’t change the template settings, but save them as “new name” (put in appropriate name) before making any changes. 4. Sequence set up: a. only use the line number, identifier 1, date and time options for folder and filenames. DO NOT use the method name. b. since identifier 1 is used in the naming of the file or folder, is can only contain letters, numbers, dash, underline, and single spaces. DO NOT use any other symbol. c. the method you use will now be found under:

<P.I. name >\method name d. the export templates are found under: finnigan\stuff\export\<P.I.name>\ e. the folder name settings should not have any thing checked in the “pre” section, “date” or “date and time” in the “post” section, and the folder name should read: <P.I. name>\<your name>\<anything else you want> f. the file name settings should have “line” and “identifier 1” checked in the “pre” section, then “date” and “time” in the post section, with the middle section empty (delete the “Acquisition” or “ACQ results” that tends to pop up into that box). g. if you are exporting your data to an excel file, the export folder name can be left blank and an “export” folder will appear under your data folder. h. remember to do a peak centering (box is checked) for each line, otherwise the time will not reset itself between runs. i. at the end of a sequence, please put the instrument in standby mode, that is run the standby method on the last line (no peak centering needed for this line). Please do not check any other option in the start dialog/option box, as they are not working yet. 5. Log in to log book for your instrument and put a print out of your sequence in the folder (three hold punch can be found in the mass spec room). 6. Load your samples, set the gases, check gas pressures on the tanks, let the system purge as needed, power up mass spec (select appropriate gas), check backgrounds as needed, and set system to remote control as needed (e.g. Costech EA). 7. If all ok and set up, push start and ok. 8. Wait for the first run to start to make sure all is ok and also check it at end of first run (in case Costech EA is not in remote mode or the GC is not set to auto-prep run, etc.)



9. Please NOTE: If the instrument and/or peripheral has not been used in a while, it is a very good idea to come down a day or two before you want to start your run with some junk samples so we can run some conditioning runs and make sure everything is behaving well. 10. At the end of a run, please take your print outs, e-mail or transfer the files from here to yourself (data and/or export files) so you have a copy of the data incase something happens down here, and can reprocess data if need be. Please do this before someone else starts their runs.



Using the Costech EA on the Delta Advantage (oxidation: N, C analysis): 1. Sample Preparation: a. Sample i. Size. The instrument can detect down to ~1 umole gas, i.e. 1 umole C as CO2 and 2 umole N as N2. However you should use at least twice as much sample as that for consistent and accurate results--signals should be greater than 2000 mV. For N and C analysis, the limiting material is usually the N. The carbon is usually in excess and the CO2 peak coming off the EA can be diluted to bring that signal on scale. Usually we run at 2-3 mg for plant and 1 mg for animal samples, with the nitrogen being the limiting factor and very low %’s of nitrogen samples require more sample. Please note, if you are just running a sample for carbon analysis, you usually can use much less and not dilute the CO2 peak. It is always best to run a series of test samples to find the best amounts and settings for your material, as well as to check for sample homogeneity. ii. Preparation. Samples should be clean, unless you’re running dirt samples, dried and homogenized. If you samples have carbonate deposites on/in them, e.g. marine sediments, that will show up in the 13C value for the CO2. You’ll have to get rid of them, e.g. HCl fumigation. Please read up on the literature for your sample type so you can apply the proper protocols. If you use filter samples, you should roast and acid wash the glass/quartz filters before using them. You may have to add a burning aid, e.g. extra tin strips or powder. Also, if you use filter samples, you’ll have to make up a filter standard curve series for data correction. iii. Type. Plants. Animals Filters and Sediments. See me before running filter samples as they require special inserts. Marine. Other. b. Standard samples and blank runs: There are three main types of runs in a sequence: sample, reference (for percent composition, reference, start reference mean, add reference mean, start reference regression and add reference regression), and blank (subtracts out areas found in blank runs from succeeding samples to give “blk corrected” isotopic ratios, blank, start blank mean, and add blank mean). You also need to run an isotopic ratio standard (run as “sample” type) every 10 samples or so, which may or may not be the same material you use for the percent composition reference. The best reference is something that is close in composition and size to your sample. The blank should be a clean capsule folded up like the sample capsules. If you are running filter samples, then a clean filter should be placed in the blank capsule as well. Please note, the first run in a sequence is a junk run used to condition and clean instrument and may have a large blank signal. If you’re pushing the limits of the instrument, high or low, or filter samples, then you’ll need to run a standard curve, and use it to apply corrections to your data. The autosampler has 49 usable positions, and with one junk, one blank, three references (different sizes), and five standards, you have approximately 38 slots available for your samples which you may want to run in duplicate or triplicate. Please see the discussion on setting up a sequence below. 2. Computer: a. Wake up the computer, i.e. move mouse or hit the “shift” key, log in if necessary (ID and

password available from staff). b. Access Isodat:

i. Isodat’s Instrument Control and Isodat Acquisition (spelled Aquisition) programs should already be running--with their buttons on the bottom button bar.

ii. If there is a small red square on the right side of the bottom button bar, that means Acquisition is acquiring data--the instrument is already in use--do not use it.

iii. If Isodat is not running, from the “Start” menu or the Isodat 2.0 folder on the desktop, start “Instrument Control” and then “Acquisition” by clicking on the icons Under instrument control, power up the mass spec’s source (click on the red sunburst on the menu



bar in the “accessories” panel on the top left side of the Instrument Control window) and turn on the source heater (click on button to left of “source heater” in the MS State panel--on is “green”).

3. Mass Spec: a. Analyzer pumps: The analyzer pump button is on: i.e. it is pushed in and the red indicator light on that button is on (inlet pumps are off since we don’t have any). Get help if the analyzer pumps are off. b. Panel lights: NO red lights should be lit--only green ones, with the “Inlet pumps” section dark (since we don’t have any inlet pumps). c. Panel lights, bottom section: The bottom two lights should be lit if the source is on. If they are off the can be turned on via the computer--Isodat Instrument Control, “Accessories” panel, click on red sunburst to turn on source, grey sun burst turns source off, and “lightning” icon toggles the high voltage on and off. Sometimes you may have to turn off the source then turn it back on to get it to work properly. 4. Check Instrument and Gas Configuration in Isodat: a. Configurations: in Isodat Acquisition, check the bottom left hand side of window for the following icons with the settings in the adjacent box: i. Toolbox icon: select “Conflo” if it is not already picked. ii. The gas icon (horn with bubbles coming out of it--hey, I didn’t design it): select “N2”, it should have been in “standby” mode. b. Panel Settings: “MS” panel on left side of Isodat Acquisition window, top: High Voltage= 2.949 Magnet=896 HV= 3.04 (Not 0.3--click on “lightning” icon to turn HV back on if it’s at 0.3) Box ~0.5 Trap ~0.5 (may vary with current settings) Vac ~1.4 x 10-6 mBar Mass listed for N2: 28 29 30 The mV readings for the different masses may be high at this time, depending on Conflo and Costech EA set up. 5. Check Peripheral--Costech EA, Conflo III, and Gas tanks a. Costech EA: The EA should be in ready mode, i.e. the three LED’s for the right and left furnaces and the GC column should be green. If they are red (and blinking) with “stdby” listed in the EA’s display, then it is in standby mode. Please note i. If it is in standby mode, press the “work” button then “enter” to go to work mode--this heats up the furnaces and starts the gases, He flowing. You will have to wait until the system is warmed up, all LED’s green and not blinking, before you’ll be able to start a run. In the mean time, continue with sample setup and loading. ii. Open up the O2 flow (using the O2 regulator, adjust to mark on gauge. Do not adjust bottom flow regulator knob, it should always be open to the correct setting) Select sample size (amount of O2 needed to burn sample): micro, semi-micro or macro. When in doubt, go with semi-micro. Generally, 3.5x5 cups are micro, 4x6 micro or semi-micro, 5x9 cups are semi-micro, and larger sizes are macro. Currently we are using a 12 sec drop delay for all samples sizes, but that could change. If it is in remote mode, REM is on the display, you have to hit the “abort” key, then “enter”, adjust the O2 size, then put it back into remote mode by hitting the “remote” key and then “enter”. Exercise the valves--that is do a dummy run (or two) before loading samples to get rid of any trapped air. You can do this from Isodat if the Costech EA is in remote mode. On the left hand side in the acquisition module (or instrument control module), go to the “Conflo II/III interface” box,



and click the radio button and it will turn green. Click it once more to turn it off, back to gray. That will start a one minute “run” on the EA. iii. Check that the “ash collector” quartz insert has enough room left in it for your run--otherwise it needs to be replaced--this is dependent on your samples (larger create more ash, smaller ones less ash--approximately 300 3.5x5 or 200 4x6 or 150 5x9 cups per insert. If the furnace looks bright orange, if should be ok. If there is a dark crust above the bright orange glow, the insert probably needs to be replaced. When you go to load samples, stick in dipstick to see if it needs to be replaced--remember that its still hot so don’t grab the end of the the dip stick or set it down on something that is not heat resistant. Filter samples are a special case and require a special insert and increase delay between runs. Basically the glass filters melt and will ooze out the vent holes in the standard ash collector, and then we may have to replace the entire reactor. Therefore, we put a second insert in the insert (a quartz tube) to protect the original insert’s vents. We usually have to swap the collector out after one filter sequence, and always before reverting back to standard runs. b. Conflo III gas settings: Check the He and reference gas settings, adjust if needed. DO NOT change the He gas setting on the Costech EA, only on the Conflo III: i. He: top knob and gauge, for sample dilution. It also dilutes the reference gases, so higher pressure means lower signals. A minimum pressure of ~8 psi (>0.5 bar) is needed to keep the atmosphere from bleeding into the open split and ultimately into the mass spec. If you are diluting the CO2 runs in your samples, try running at 25 psi for 3 mg samples, 15-20 for 2 mg, and 10-15 for 1 mg, assuming they’re roughly 50% C by mass. Please note, if you ‘re looking C only on small samples, that is you will not be diluting the CO2, keep the He flow at 8-10 psi. ii. Ref1: middle knob and gauge, CO2. After He pressure is set, adjust CO2 pressure accordingly. If you switch the mass spec to the CO2 gas configuration, you can monitor the signal intensity of CO2 and adjust the pressure accordingly (from 5 to 35 psi). In the Conflo panel in Isodat Acquisition, click on the He-dilution tube in the drawing to lower it (blue line from He-dilution section seen in open split diagram) then click on the Ref1 line to lower it too (also blue line extends from labeled ref1 line). Wait ~10 sec to gases to get into mass spec then wait for signal to stabilize. Adjust gas pressure within ranges to get desired intensity for the reference peak. Depending on the He pressure, the maximal intensity ranges from 20,000 mV (8 psi He, 35 psi CO2) to 6900 (20 psi He, 35 psi CO2), with lower values expected at higher He pressures. If your samples are consistently off from the standard gas peak, check the values of the standard runs and correct the sample values. You can expect the isotopic values to drift up to 0.1 per mil per 1000 mV difference in signal intensity. For samples with dilution on for the CO2, reference 1 is usually around 30 psi iii. Ref2: bottom knob and guage, N2. Treat like reference one, however raise ref1 up in the open split and lower Ref2 (nitrogen), switch to “N2” in the gas configuration, and adjust Ref2 to the desired intensity, like what was done with Ref1 above. Please note, at the same pressures, the intensity of N2 while in N2 mode is about half that of the corresponding signal of CO2 in CO2 mode. Also note, in N2 mode, there is a relatively high mass “30” background. This is due to NO formation in the source as the ionized N2 reacts with residual water or other oxygen containing species in the source. This is usually around 25 psi when nitrogen samples are being run. c. Check main He, Reference and O2 tanks: Gas tanks behind Costech EA, tank pressures should be greater than 500 psi. The He and N2 tanks start at ~2000psi at the tank, the CO2 is steady at ~800 psi. The feed pressure should be ~50 to 60 psi (only He feed to Trace GC on 253 higher at ~80 psi). The O2 tank is on wall next to entrance to 078e (Costech room) and feeds EA and GCC. Replacements should be ordered or here if tanks are below 500 psi, and they should be replaced if they fall below 300 psi. 6. Set-up Run a. Open Sequence template in Isodat Acquisition (or Workspace) module, then immediately save it in your sequence folder as your current sequence with an appropriate name. b. Fill out sequence:



i. Remember, an initial junk or blank run is needed to condition the system. This is not to be used as your official blank for the series. ii. Run one blank, after the “junk” sample at the start of a sequence (sample type “blank”, or you can average two or more blanks by using “start blank mean” then “add blank mean” types. The areas of the peaks detected (if any) will be subtracted out of corresponding peaks in subsequent runs with the corrected results listed under “blk corrected.” Uncorrected results are also listed. The main source of the blank signal is N2 contamination of the O2 gas stream. It is small but very heavy (150 to 200 per mil vs air), and you may have to adjust your peak detection setting to pick it up. You need to correct for it. iii. Run at least one “reference” type run for percent composition immediately after the “blank” runs. Reference runs can be run as a single “reference” type run, as an average of multiple runs (types “start reference mean” and “add reference mean”), or as a standard line covering a range of sample sizes (types: “start reference regression” and “add reference regression”). Usually two or three references at different sample sizes are used. A typical compound used would be acetanilide with 10.36% N and 71.09% C, with the amounts used adjusted to bring the total amount of N and C in line with your sample. You can also use material closer in composition to your samples, e.g. cabbage or trout. Please note, the percent composition of your reference material is part of your method, and you will need to edit your method if you change reference material. The “reference” sample is only for percent composition, but the material you use can double as a house isotope ratio standard. iv. Run isotope ratio standards (house reference material), run as “sample” types, every 10 samples or so. Please note, there are only 49 slots in the autosampler, with ca. five used for junk, blank and percent composition runs, leaving 44 available. With four used for reference samples, forty are left for unknown samples. If you run in duplicate, that means 20 different samples can be run per autosampler load. v. If possible, group samples by isotopic ratio in a series and is it best to run different types of material, e.g. plant or animal, in different runs (or with different methods and blank and reference in same sequence). Depending on sample size, there is a negligible memory effect (less than 0.2%), which is not seen if samples are close in composition, e.g. if there is a 5 per mil jump between samples, the memory effect is at most 0.01 per mil. For a given series and size of samples, triplicate runs of known samples with differing isotopic ratios can be used to calculate the memory effect and then the corresponding correction applied to the data. vi. Fill out rest of sequence: peak center: check for all samples except last “standby” mode run. amounts: amount, in mg, of sample (not tin or filter). type: as discussed above--a blank and reference needed for every series. port: autosampler slot. Start at position next to furnace opening. Identifier1: used in filename, only letters, numbers, dash and space allowed. preparation: additional information, e.g. gas pressure settings, sample prep. etc. method: should already be in your template, “PI name\method name” last run is “standby” method. other columns, e.g. “identifier2”, “comments”, available if needed c. Load Autosampler: i. Have samples ready. The weighed out samples in the folded tins are usually stored and transported in a 96 well plate. The position of a given sample in the plate can be noted in the samples identifier or preparation column in the sequence, and a printed out copy of the sequence can serve as a loading guide. ii. Open autosampler: Check He pressure on EA. Open center valve to release gas pressure, then unscrew knurled nuts to loosen fasteners. Let fasteners drop off to sides and open top cover. iii. Check autosampler for previous samples (sometimes they stick).



iv. Load in your samples v. Remove any dust on o-ring seal (usually tin oxide ash, will not interfere with samples), and close cover. vi. Re-fasten cover--start with front fastener. Tighten almost metal to metal vii. Let system purge for ~5 min., then close center pressure relief valve. Please remember to do this, otherwise the tank will bleed out. Check He pressure on EA--if it doesn’t come back up to previous setting, you may be leaking He out the autosampler. Release pressure on autosampler, tighten fasteners, then close pressure release valve. Occasionally, you’ll have to reopen cover, wipe the seals, close and purge again or even remove pressure release valve, clean it and put it back into place. 7. Start Run a. Put Costech EA in to Remote mode so it can communicate with the mass spec, if needed. Push “remote” then “enter” on keypads, “REM” appears on LCD display. b. Wait for systems to stabilize. i. All lights on Costech EA should be green now, i.e. all furnaces are at temperature. ii. Oxygen is flowing through O2 vent (this should be fine--Costech O2 pressure should be at ~ 1 bar) iii. Costech He pressure should be at mark ~1.1 bar, no Air pressure. iv. Mass spec signal, in N2 gas mode, should have stabilized, preferable <40 mV on mass 28--on good days, <30 and we won’t talk about bad days. This may vary over time. c. Double check options in Sequence. Click on the “Options” icon in the Sequence panel. This should be set up for you in the template. The folder name should be “\PI name\yourname\”. The “date” or “date and time” should be checked in the “post” selection to be appended to the listed name. If you type in a name following “...yourname\” , then the date or date and time will be appended to that name. The filename section should be blank--delete “Acquisition” if it appears”. The file name consists of the line number and identifier 1 in the “pre” section followed by the “data and time” in the post section. If you type a name in the box, it will fit in between those two sections. Printout and export options can also be adjusted at this time. If want to save the changes you’ve made, click on “ok” else “cancel”. d. Save Sequence, Start Run. Save sequence file, then click on green arrow icon in sequence panel to start run. Basically, the “options” page opens up again, but this time “Start Sequence” appears in the top (blue) part of the window. If it’s ok, hit “ok”. 8. Monitor first sample, then check back when a your first real sample is running. Follow to first run to make sure it is running OK (this is when you’ll realize you forgot to put the EA into remote mode). Check back later to make sure standards ran OK and then watch your first sample or two to make sure they’re fine too--this is especially important for new types of samples. If something is wrong, e.g. standards are off or it turns out the sample sizes are too large (or small), click on the red “stop” button to abort the sequence. It will ask you if you want to finish current sample--answer “yes” if the sample has dropped already. 9. End of Run--shut down:

a. gas configuration should be in standby mode and conflo dilution on (last line of sequence should have done this) b. On conflo, He presure should be at least 8psi and up to 16 psi, reference gas pressures should be around 5 psi. Please note, reference gases take some time to bleed out so don’t keep turning the knob--one full (counter clockwise) turn drops the pressure about 5 psi, so only use 5 full turns to go from 30 psi to 5 psi, then let the pressure drop on its own. c. save your data, reprocess if need be and copy your data to your computer. d. please note, we are NOT putting the Costech itself into standby anymore



Using the Thermo TC/EA on the Delta XP (pyrolysis/C-reduction: H, O analysis): 1. Sample Preparation: a. Samples. i. Sizes. The instrument can detect down to ~2 umole gas, i.e. 2 umole O as CO and 4 umole H as H2. However you should use at least twice as much sample as that for consistent and accurate results--signals should be greater than 2000 mV. For H and O analysis, the limiting material will depend on the sample type. If there are significant differences between amounts of H and O, weigh out enough material to accommodate the limiting factor, then set the method to dilute the other one if needed. It is always best to run a series of test samples to find the best amounts and settings for your material, as well as for to check for sample homogeneity. ii. Types. Phosphates and Sulfates as silver phosphate and barite for 18O analysis. These samples are usually dried after isolation by heating up to 500oC under vacuum on our vacuum lines. They run fairly cleanly at ~200ug/run, and can be run with added H2 in the carrier gas stream (use the Aux gas on the TC/EA) to maintain reducing conditions. When run neat, barites give close to 100% yields (when compared to benzoic acid), phosphates a bit less, with consistent isotopic values, though there may be slight memory effect. You cannot run phosphates samples after running barites, a new or cleaned reaction tube is required. After ~150 runs (three autosampler loads), the crucible needs to be changed. After ~300 total runs, the reaction tube needs to be changed/cleaned. Organic material. Unless you’re interested in trapped/absorbed water, these must be dried before being run. The drying is sample dependent. The IAEA/NBS polyethylene foil can be run neat--it doesn’t trap water. Pure amino acids samples may crystallize out as hydrates, and that water would need to be removed. Dried vs undried samples can also be run to determine value of the trapped water--just remember to propagate the error. These samples tend to leave a fair amount of residue behind, and the crucible has to be swapped out after every autosampler run, ~50 samples for plant material--though certain materials swamp the reaction column much sooner (<30 samples). This also depends on sample size and homogeneity, and amounts can range from 200ug to >1 mg depending on actual samples. Remember, the hydrogens on -OH and -NH2 groups are exchangeable and the samples have to be dry for good results. Nitrates as dried silver nitrate for 18O analysis. Dry samples, ~200ug, can be run at slower than normal carrier gas flow rates and lower GC temperatures. Nitrates will breakdown in the reaction tube to N2 and CO, though you can generate some CO2 if flow rates are too high. The lower GC temperature is needed to give clean separation between N2 and CO2. You may be able to analyze both the N2 and the CO for 15N and 18O together, though that has proved to have some problems here. The N2 peak can interfere directly with the CO peak, and even if there is baseline separation for the main peaks, N2 can form NO in the source that does not pump away quickly, and it will interfere with 18O determination. The work around is to use small samples, dilute the N2 peak as it elutes to minimize NO formation, then re-run the same sample for N2 analysis (EA or TC/EA). For the 18O analysis, H2 in the carrier gas stream can help (i.e. use the Aux gas for the carrier gas on the TC/EA). Crucible is cleaned every 150 samples, reaction column every 300. Carbonates. We use the GasBench, but you’re welcome to give it a try. Hydrous Minerals run neat for 2H analysis. Dry samples, amount can be >2mg depending on quantity of -OH in sample, and 18O analysis can also be done at the same time. This will give total labile H and O, which should be all the H but only a fraction of the O, depending on the mineral. Silica and alumina do not freely release their O under standard conditions. Other types of minerals may or may not release all or part of their O, and there may be isotopic exchange between oxygens at 1450oC prior to release. Crucible is cleaned after ~50 samples, depending on amount of material needed per run. Silicates, and other materials run with additives. Studies have shown that you can look at silicate O by adding either KF and/or -C2F4- (ptfe, aka Teflon) to the sample. That fluorinates the silicate, releasing the O that then forms CO in the TC/EA pyrolysis/carbon reduction tube. Care must be taken to trap any reactive compounds, especially HF, before the cold trap. The gas coming out of the TC/EA reaction furnace passes through a chemical trap: ascarite (sodium



hydroxide on vermiculite, an acid and CO2 trap) and magnesium perchlorate (water trap) loaded into a quartz tube (6mm OD, 35-40 cm long, or larger). Sodium bisulfite (halogen trap) may also be added before the ascarite. The chemical trap is then connected to 1/8” stainless steel tubing, looped back on itself, for the cold trap (liq N2), which is then connected to the switching valve--see discussion below. The cold trap itself is there to keep fluorinated material, e.g. SiF6, CxFy, etc., from making it to the mass spec Typically we use 100-200ug silicate, 300-400 ug KF or 1-1.5mg PTFE, and run at a reduced flow rate. Remember, the KF is hydroscopic so work in a dry box if possible. Also, the PTFE + KF combo seem to work better than either alone. The sequence starts with blank (empty capsule) and reference (benzoic acid) runs, followed by a couple of conditioning/clean up runs (KF or PTFE alone), a fluorinated blank (KF or PTFE alone), then the samples. Please note, each sample usually requires one or two clean up runs (KF or PTFE alone) afterwards, and even then you may still see a memory effect. Fluoridated graphite (powder--don’t breath it in) can be used instead of PTFE, but it is much easier to slice off a fixed size of teflon tubing we have and place that into the silver capsule. Both PTFE and the fluoridated graphite are permeable to O2 from the air, which adds to the background. The KF must be dry. These runs should be done in a dedicated reaction tube, and a freshly packed tube will require extra conditioning/clean up runs. The crucible will have to be replaced after each PTFE sequence (49 runs) and every other KF sequence--tube cleaning with every second crucible change. Attempts to run at temperatures <1450 using PTFE did not do well. Initial tests combining KF with PTFE in a single run (say 200-400 ug each), seem to indicate they’ll work well together, as long as the KF is dry. Hydroscopic additives, KF, AgF2, etc., are difficult to work with without a dry box. Liquids in capsules. “Cold weld” device available. Load sample into smooth wall silver cup and seal in device--then treat as a solid sample. Liquids injected via PAL autosampler. Available with some modifications of TC/EA set up. The graphite cone that sits on top of glassy C tube in reactor is replaced by a piece of glassy C tube that brings the total height of the glassy C tubing almost to the top of the alumina tube. A ball of silver wool in placed on top of this, and the injection port is sealed on top of reactor--in place of the zero blank autosampler. A 500 nL syringe (SGE, plunger in needle) is mounted on the CTC-Pal autosampler. Move the zero-blank autosampler out of the way, copy tray 5 to tray 1 so you’ll use the small sample tray, attach the “wash and waste” tray and double check positions of injection port, sampler tray and “waste” tray. Use autosampler method 2, which is for 500 nL SGE syringes, injects 400 nL, and havs the needle wash protocol (5-10x with DI water, 2-3x with acetone that is injected to waste vial that is under vacuum). This gives both DH and 18O results, but with larger error than the H-device and CO2-water equilibration. Expect +/- 2 per mil for H and +/-0.3 per mil for O on good days. Please note, this should give values for the total H and total O in the sample, so it probably would not be the way to follow exchange reactions with a labeled solute. b. Standard samples and blank runs: There are three main types of runs in a sequence: sample, reference (for percent composition, reference, start reference mean, add reference mean, start reference regression and add reference regression), and blank (subtracts out areas found in blank runs from succeeding samples to give “blk corrected” isotopic ratios, blank, start blank mean, and add blank mean). You also need to run an isotopic ratio standard (run as “sample” type) in the middle and/or at the end of a sequence, which may or may not be the same material you use for the percent composition reference, to account for any drift during the sequence. The best reference is something that is close in composition and size to your sample. The blank should be a clean capsule folded up like the sample capsules. Please note, the first run in a sequence is a junk run used to condition and clean instrument. The autosampler (solid samples) has 49 usable positions, and with one junk, two blanks, two references, and six to eight standards, you have approximately 37 slots available for your samples which you may want to run in duplicate or triplicate. Please see the discussion on setting up a sequence below. Also, note the special situations mentioned under sample preparation, e.g. silicate runs.



2. Computer: a. Wake up the computer, i.e. move mouse or hit the “shift” key, log in if necessary (ID and

password available from staff). b. Access Isodat:

i. Isodat’s Instrument Control and Isodat Acquisition (spelled “Aquisition”) programs should already be running--with their buttons on the bottom button bar.


iii. If Isodat is not running, open the Isodat 2.0 folder on the desktop and start “Instrument Control” and then “Acquisition” by double clicking on the icons. These can also be found under the “start” button. Under instrument control, power up the mass spec’s source (click on the red sunburst on the menu bar in the “accessories” panel on the top left side of the Instrument Control window) and turn on the source heater (click on button to left of “source heater” in the MS State panel--on is “green”).

3. Mass Spec, XP: a. Analyzer and Inlet pumps: The analyzer pump and inlet pump buttons are on: i.e. it is pushed in and the red indicator light on that button is on. Get help if the analyzer pumps are off. b. Panel lights: NO red lights should be lit--only green ones, c. Panel lights, bottom section: The bottom two lights should be lit if the source is on. If they are off the can be turned on via the computer--Isodat Instrument Control, “Accessories” panel, click on red sunburst to turn on source, grey sun burst turns source off, and “lightning” icon toggles the high voltage on and off. Sometimes you may have to turn off the source then turn it back on to get it to work properly. 4. Check Instrument and Gas Configuration in Isodat: a. Configurations: in Isodat Acquisition, check the bottom left hand side of window for the following icons with the settings in the adjacent box: i. Toolbox icon: select “TC/EA” if it is not already picked. ii. The gas icon (horn with bubbles coming out of it--hey, I didn’t design it): select “co”, it should have been in “standby” mode. b. Panel Settings: “MS” panel on left side of Isodat Acquisition window, top: High Voltage= 2.949 Magnet=896 HV= 3.04 (Not 0.3--click on “lightning” icon to turn HV back on if it’s at 0.3) Box ~0.5 Trap ~0.5 (may vary with current settings) Vac ~1.4 x 10-6 mBar Mass listed for N2: 28 29 30 The mV readings for the different masses may be high at this time, depending on Conflo and TC/EA set up. To double check to see if the conflo is opened up to the mass spec, pulse in Ref 1, CO, for a few seconds and see if the mass 28, 29 and 30 signals respond appropriately. Please contact the staff if you have to switch the mass spec to the conflo. 5. Check Peripheral--Thermo TC/EA, Conflo III, and Gas tanks a. Thermo TC/EA: The TC/EA should be the peripheral opened up to the mass spectrometer, and be the active peripheral in Isodat.

i. The reactor needs to be brought up to temperature and the GC temperature lowered to the working range. The reactor is usually set to 1450, the GC to 90 from their respective standby temperatures of 900 and 125. Press the “P” button twice on the appropriate controller, use the arrow keys to set the new temperature, the push “P” again. The ramp rate,



how fast it gets to the new temperature, should be set to 5oC per minute for the reactor, so it will take some time to get to the new temperature.

ii. The gas four-way switching valve for the cold trap, on top back of TC/EA should be pointing towards the wall, and the three way valve on the vent line should be pointing towards the capillary tubing to restrict flow. This way, the gas flowing out of the TC/EA and through the cold trap is directed into the GC column, and not out the vent line.

iii. TC/EA Gas pressures. Purge gas, He, should be about the same as the He gas line, usually 21 psi, but can range from 15-22 psi. Aux gas pressure, if it is not being used, will read the same as the He gas pressure. If you are running with the Aux gas instead of He, first lower the He gas pressure to it is running ~10psi lower than the desired pressure. Now turn up the Aux gas pressure until it brings up the reading on the He gauge to the desired reading. The Aux gas pressure itself will run about 10 psi higher than the He pressure when it is the gas source.

iv. Close valve on Costech’s Zero-Blank autosampler and open valve on purge gas line. Please do not forget to reopen valve and close purge gas line after samples have been loaded and flushed and before running samples (see below)

b. Conflo III gas settings: Check the He and reference gas settings, adjust if needed. Please note, an exhaust hose is mounted on the top of the Conflo to remove the H2 and CO gases, make sure it is in place. Also, at the start of the day, you have to run an H3+ factor correction routine (see Ref2 below) if you are doing 2H analyses. i. He: top knob and gauge, for sample dilution. It also dilutes the reference gases, so higher pressure means lower signals. A minimum pressure of ~8 psi (>0.5 bar) is needed to keep the atmosphere from bleeding into the open split and ultimately into the mass spec. ii. Ref1: middle knob and gauge, CO. CO is a dangerous gas, IF ALARM SOUNDS, RUN AWAY!!! After He pressure is set, adjust CO pressure accordingly. If you switch the mass spec to the CO gas configuration, you can monitor the signal intensity of CO and adjust the pressure accordingly (from 5 to 35 psi). In the Conflo panel in Isodat Acquisition, click on the He-dilution tube in the drawing to lower it (blue line from He-dilution section seen in open split diagram) then click on the Ref1 line to lower it too (also blue line extends from labeled ref1 line). Wait ~10 sec to gases to get into mass spec then wait for signal to stabilize. Adjust gas pressure within ranges to get desired intensity for the reference peak. Depending on the He pressure, the maximal intensity ranges from ___ mV (8 psi He, 35 psi CO) to ___ (20 psi He, 35 psi CO), with lower values expected at higher He pressures. If your samples are consistently off from the standard gas peak, check the values of the standard runs and correct the sample values. You can expect the isotopic values to drift up to 0.1 per mil per 1000 mV difference in signal intensity. iii. Ref2: bottom knob and guage, H2. H3+ factor

Raise ref1 up in the open split and lower Ref2 (hydrogen), switch to “H2” in the gas configuration, and adjust Ref2 to ~10psi. Run H3-factor routine (standard on-off for Ref2 in H2 mode). After every third pulse, raise Ref2 pressure by 5 psi until you get to 35 psi. The pulse ends when the Ref2 line is withdrawn, as shown on conflo diagram--you do not have to wait for actual peak in chromatogram to finish. Once done, click on the H3+ icon on left side of screen, and select “Top cf document”, then OK. The program will calculate the H3+ factor and it should be close to the previous value (well within 1). Choose that value to use in your runs.

One the H3+ factor has been calculated, lower Ref2 again, wait a few seconds until the signal comes up, then set it to the desired intensity, like what was done with Ref1 above. Please note, when lowering the pressure to lower the signal, the gas will take some time to bleed out. It it usually better to go below the desired value then raise the pressure up to bring the value up to the desired reading. c. Check main He, Reference and auxillary (H2 in He) tanks: Gas tanks nest to the computer, tank pressures should be greater than 500 psi. The He, H2 and CO tanks start at ~2000psi at the tank. The feed pressure should be ~50 to 60 psi (only He feed to Trace GC on 253, not here, higher at ~80 psi). d. Autosampler:



6. Set-up Run a. Open Sequence template in Isodat Acquisition (or Workspace) module, then immediately save it in your sequence folder as your current sequence with an appropriate name. b. Fill out sequence: i. Remember, an initial junk or blank run is needed to condition the system. This is not to be used as your official blank for the series. ii. Run one or two junks at the start of a sequence (sample type “sample). The silver capsules are usually clean, so no blanks are needed--but always double check a new batch or supplier. However, if you are using additives, e.g. KF or teflon, then two or three junks, capsules with additives but no sample, are need to condition/clean the furnace and one or two blanks, also capsules with additives, should be run. The areas of the peaks detected (if any) will be subtracted out of corresponding peaks in subsequent runs with the corrected results listed under “blk corrected.” Uncorrected results are also listed. If the blank is small, you may have to adjust your peak detection settings to pick it up. You need check it to see if you need to correct for it. iii. Run at least one “reference” type run for percent composition immediately after the “blank” runs. Reference runs can be run as a single “reference” type run, as an average of multiple runs (types “start reference mean” and “add reference mean”), or as a standard line covering a range of sample sizes (types: “start reference regression” and “add reference regression”). A typical compound used would be benzoic acid with 10.36% H and 71.09% O, with the amounts used adjusted to bring the total amount of H and O in line with your samples. Please note, the percent composition of your reference material is part of your method, and you will need to edit your method if you change reference material. The “reference” sample is only for percent composition, but the material you use can double as a house isotope ratio standard. iv. Run isotope ratio standards (house reference material): run as “sample” types, in triplicate or quadruplicate like your samples. You should run at least two if not three different references, preferably bracketing the isotopic ratio of the samples, and one should be re-run atht end of a sequence to check for drift. Please note, there are only 49 slots in the autosampler, with ca. five used for junk, blanks and percent composition runs, leaving 44 available. With twelve (4x3) used for reference samples, thirty two are left for unknown samples. If you run in quadruplicate, that means 8 different samples can be run per autosampler load. v. If possible, group samples by isotopic ratio in a series and you need to run samples of the same type together. Depending on sample size and type, there can be memory effect. For a given series and size of samples, triplicate runs of known samples with differing isotopic ratios can be used to calculate the memory effect and then the corresponding correction applied to the data. Alternatively, discard the first run in the group and average the latter two only. vi. Fill out rest of sequence: peak center: check for all samples except last “standby” mode run. amounts: amount, in ug, of sample (not capsule). type: as discussed above--sample. blank or reference (amount% only) port: autosampler slot. Start at position next to furnace opening. Identifier1: used in filename, only letters, numbers, dash and space allowed. preparation: additional information, e.g. gas pressure settings, sample prep. etc. method: should already be in your template, “PI name\method name” last run is “standby” method. other columns, e.g. “identifier2”, “comments”, available if needed c. Load Autosampler: i. Have samples ready. The weighed out samples in the folded tins are usually stored and transported in a 96 well plate. The position of a given sample in the plate can be noted in the samples identifier or preparation column in the sequence, and a printed out copy of the sequence can serve as a loading guide.



ii. Close furnace, open purge line then open autosampler. Open center valve to release gas pressure, then unscrew knurled nuts to loosen fasteners. Let fasteners drop off to sides and open top cover. iii. Check autosampler for previous samples (sometimes they stick). iv. Load in your samples v. Remove any dust on o-ring seal (usually “charcoal”, will not interfere with samples), and close cover. vi. Re-fasten cover--start with front fastener. Tighten almost metal to metal vii. Let system purge for ~5 min., then close center pressure relief valve. Please remember to do this, otherwise the tank will bleed out. Close purge gas valve and open autosampler up to the furnace. 7. Start Run a. Put LN trap on--make sure TC/EA flowing to GC. On back of system, switching valve should be pointing towards the GC (back wall) so the TC/EA is flowing to GC and not being vented. Loops coming off TC/EA go into cold trap (dewar, Liquid N2--though a dry ice trap would work too). Please note, if you running with fluorinated additives/samples, you should have an acid/halogen trap already in-line before the cold trap. b. Wait for system to stabilize. i. TC/EA’s furance and GC temps should be stable and holding at the set values. ii. Double check gas pressures. Aux. gas should be reading same as He if it’s not being used as the gas source. If the Aux gas is the source of the gas for your TC/EA runs, then it should read ~10psi higher than the He gas pressure reading. iii. Mass spec signal, in CO gas mode, should have stabilized, preferable <400 mV on mass 28. This may vary over time. Older reaction tubes tend to take longer to bake out have higher backgrounds. c. Double check options in Sequence. Click on the “Options” icon in the Sequence panel. This should be set up for you in the template. The folder name should be “\PI name\yourname\”. The “date” or “date and time” should be checked in the “post” selection to be appended to the listed name. If you type in a name following “...yourname\” , then the date or date and time will be appended to that name. The filename section should be blank--delete “Acquisition” if it appears”. The file name consists of the line number and identifier 1 in the “pre” section followed by the “data and time” in the post section. If you type a name in the box, it will fit in between those two sections. Printout and export options can also be adjusted at this time. If want to save the changes you’ve made, click on “ok” else “cancel”. d. Save Sequence, Start Run. Save sequence file, then click on green arrow icon in sequence panel to start run. Basically, the “options” page opens up again, but this time “Start Sequence” appears in the top (blue) part of the window. If it’s ok, hit “ok”. 8. Monitor first sample, then check back when a your first real sample is running. Follow to first run to make sure it is running OK (this is when you’ll realize you forgot to put the EA into remote mode). Check back later to make sure standards ran OK and then watch your first sample or two to make sure they’re fine too--this is especially important for new types of samples. If something is wrong, e.g. standards are off or it turns out the sample sizes are too large (or small), click on the red “stop” button to abort the sequence. It will ask you if you want to finish current sample--answer “yes” if the sample has dropped already. 9. End of Run--shut down:

a. gas configuration should be in standby mode and conflo dilution on (last line of sequence should have done this)



b. On conflo, He presure should be at least 8psi and up to 16 psi, reference gas pressures should be around 5 psi. Please note, reference gases take some time to bleed out so don’t keep turning the knob--one full (counter clockwise) turn drops the pressure about 5 psi, so only use 5 full turns to go from 30 psi to 5 psi, then let the pressure drop on its own. c. Use He as the carrier gas. If you were using the Aux gas as the carrier gas, switch back to using He--turn down Aux gas pressure and bring He pressure back up to 20 psi. The reading for the Aux gas gauge will track the He pressure gauge if the Aux gas is no longer the gas source. d. VENT THE COLD TRAP! Make sure purge gas pressure is up to 15 to 20 psi. Switch valve on back of TC/EA so it points away from the GC (away from wall), the purge gas is now flowing directly to the GC and the gas from the TC/EA flows through the trap and out the vent line. Remove the dewar (the cold for the cold trap) and set it aside (clean/empty as needed). Open vent line up--turn valve from capillary line to vent line. Use heat gun to help warm up the cold trap and exhaust lines. Let it vent a few minutes. After cold trap has been heated and cooled back to room temp, and vented for a few minutes, switch vent valve back to capillary side then turn switching valve back to face TC/EA’s GC (towards wall). The gas now flows from TC/EA though cold trap and to GC. e. save your data, reprocess if need be and copy your data to your computer. f. Turn temperature down on TC/EA reaction furnace--to 900 oC. The GC should be set up to 125 oC

Using the Gasbench on the Delta XP: 1. Sample Preparation: a. Samples. i. Size. For headspace analysis, the instrument can detect down to ~0.5 umole CO2 in 12mL Laco Exetainer tube using the 100 uL sample loop (concentration in sample’s headspace: 50 uM or 1 ppt). If the sample is in air, not He, then the “air” peak must be diluted. Larger CO2 samples themselves can be diluted (~10x) to bring them on scale. Other gases can also be analyzed this way, including H2, N2, O2, etc. They just have to be able to be separated from any interfering gases by adjusting the GC parameters, and the Mass Spectrometer has to be tuned for them. The detection limits will vary for different gases. Other options to improve sensitivity include using a larger sample loop and adjusting the dilution settings if needed, and “freezing out” the gas of interest (e.g. CO2) with the automated cold trap (connected only when needed).

ii. Types: Air and Breath analysis (CO2 in Air). Fill cleaned Exetainer with sample of interest--blowing into tube, preferably using a straw, then capping it immediately works well enough for breath samples. Air samples can be in any clean tube with a pierce-able septa top that fits into our sample holder. Options include flushing a standard Laco Exetainer tube with the gas of interest via a two-hole needle (or two needles--one in, one out) or perhaps evacuating a tube then piercing the septa with a needle to draw in the sample of interest. Whatever protocol you choose or develop should be tested. Please note, the presence of water will lead to CO2-H2O oxygen exchange. The 13C number can be thrown off if a large amount of water (or any other liquid) is present unless CO2 partitioning (and fractionation) is accounted for. CO2-water equilibration, 18O of water. Flush cleaned Exetainer tubes with 0.3% CO2 in He for ~5 min. This can be done using the GasBench (Ref3 is 0.3% CO2 in He for flushing tubes). If need be, turn on valve to open gas to Ref3 from software, Isodat in GasBench mode. That is the only time you need to use Isodat for the flush-fill routine. Set



the pressure for Ref3 to 15 torr--open tank in back of room if was not on already and let it flush the lines for a few minutes before starting the routine. Load tube holder with your tubes (cleaned and capped). Secure the 0.3% CO2 in He flushing needle into left side (as you face it) of needle holder. On Autosampler control pad, from home page “add job” (F2 key). Tray=TrayGB, Start=position of first tube, Stop=position of last tube, Increment=1 (unless you are skipping positions), Count=1 (once is enough), Method=FlushFil (~6min flushing per tube). Hit start on the autosampler control pad (F1--please note, stop button on right side, along side dial--use it if there are any problems--like it tries to go to the wrong tray). If there is any problem with the “syringe” definition, it will trigger an error and give you an option to change syringe--do that. Physically, you do not change the syringe, you just redefine it in the software (choose 10000 nL). Once it starts, just make sure it is filling your tubes--wait until it goes to second tube to make sure you have them loaded in the correct order. Also double check the position of the needle during the flushing. It should be off to one (the right) side. If it is too close to the rim of the cap, it may catch it later. The needle may have to be repositioned. Once the tubes are filled with 0.3% CO2 in He, the water is injected into the labelled tube. Inject 200 to 500 uL sample per tube: flush clean syringe with sample, load sample past mark and get rid of air bubbles, push plunger to mark and touch off any drops on needle tip, inject needle into to tube at one side, inject sample, touch off needle on side of tube, remove needle. Tube will be under slight positive pressure--that’s ok. If any droplets get on top of septa, wipe them off. Place tubes in order into heating block (temp 26o C). Wait 18-24 h. Please note waiting time is for pure water. Salty or dirty samples may take longer (days) to reach equilibrium. . ALSO: Condensate may have to be shaken down (sorry, no centrifuge yet) so it’s not on the septa. Do not let any water into the sampling syringe. Dissolved Inorganic Matter runs. Put “three drops” 100% phosphoric acid into bottom of clean exetainer tube. Seal and flush with He (as is section c. just above, but use ref2 line, He, NOT the CO2 in He--do not open the ref3 valve from Isodat this time.). For automatic acid addition with out analysis, you need to run Isodat and use the “load-acid.met” method. This can also be set up to auto flush the tubes with He after acid addition. Once tubes are flushed, inject your sample (up to 700 uL) as above in section c. Mix your sample with the acid, then place tubes in order in heating block (temp 26o C). Wait 18-24 h for reaction to complete and CO2 to equilibrate. Please note, amount of acid may be sample dependent. See discussion on sample size above. ALSO: Condensate may have to be shaken down (sorry, no centrifuge yet) so it’s not on the septa. Do not let any water into the sampling syringe. Carbonates. Weigh out ~200 ug or equivalent of calcium carbonate, and load into cleaned round bottomed Exetainer tubes. Sample needs to be at the bottom of the tube to react. Flush tube with He, as in section c above, but use ref2 line, He, NOT the CO2 in He--do not open the ref3 valve from Isodat this time. Once tubes are flushed, you need to add the 100% phosphoric acid. This can be done automatically “online” during analysis runs, automatically without concurrent sample analysis, or manually “offline”. The default reaction temperature is 26 oC in the heating block. If there are no other experiments running in the block, that temperature can be changed to accommodate your samples. This should be done the night before to allow the heating block to equilibrate at the new temperature. For “automatic online analysis and acid addition” see analysis section below. For automatic acid addition with out analysis, you need to run Isodat and use the “load-acid.met” method--similiar to the section d. above, but without the He flushing. Alternatively, the acid can be added “offline”, i.e. with the samples in a separate heating block. Bring the acid to the



temperature of the block and the samples, then use a syringe to inject an appropriate amount of acid into each sample tube. Allow it to react at temperature before transferring to the Gasbench’s heating block for analysis. Reaction times and temperatures are sample dependant. Please note, you can try to react your carbonate samples at different temperatures to see evolution of CO2 from different minerals: e.g. react it at 26 oC, analyze, flush tube with He, put in heating block at 70oC, then re-analyze. You would need to work out appropriate reaction times and temperatures for your samples. Other methods can be worked out, e.g. add carbonate and dried sulfamic acid to the exetainer tube, seal, flush with He, then put in heating block at 220oC for 30 min. (H. Le and Q. Stuart-Williams, Chap 12 in Pier de Groot, ed., Handbook of Stable Isotope Analytical Techniques). Dissolved Organic Carbon. This method needs to be established--it is just a slight modification of the standard wet persulfate oxidation technique to use with the GasBench. A copper reduction tube should probably be placed between the sampling valve and the GC column, though test runs on sugars won’t need it. Please check the literature to see what’s being done. Concentrate sample, e.g. freeze dry or speed vac, if needed. Acidify sample and flush to remove DIC, e.g. as listed above for sampling, followed by purging with He. Add oxidant, e.g. persulfate and borax solution, and heat/sonicate to promote reaction. Acidify to drive off CO2 from solution. then analyze the headspace. . ALSO: Condensate may have to be shaken down (sorry, no centrifuge yet) so it’s not on the septa. Do not let any water into the sampling syringe. H2-water equilibration. This can be done, but we are not currently set up to do it. We use the H-device for DH analysis. Other gases/Dynamic headspace analysis. We have analyzed O2 directly. Other gases can be done as long as you have a source of gas to use as reference material, and can normalize the values with standards. Samples are continuously being sampled, and if there isn’t a constant source of gas, the gas concentration--and signal--falls with each sampling. Alternatively, this can also be used to monitor gas generation from reactions. iii. Controls/Standards/Data Processing: For all of the above samples, appropriate controls and data processing must be done. Your samples must be close to and preferably bracketed by standards, both in sample size and isotopic signature. The standards used should be similar in composition to your sample. The measurements made here are on the gas in the headspace. If you are interested in the source of the gas, you’ll need to appropriately correct the values obtained.

Water Samples: We have a spreadsheet that has the equations for converting the readings from CO2 equilibrated with water to the 18O for water itself. This number is very temperature dependent. You will also need to know the amount of water you added to each exetainer tube. The corrections are for pure water. High pH or high salt solutions may need further adjustments to give the 18O for the water, but that is something the user needs to work out for their particular set of runs. Once the conversion to 18O for water is complete, those numbers can then be corrected to the standards run along with the samples. This is fine for small shifts, less than a per mil, and should just be a linear correction over a relatively short range. Larger offsets may mean something is wrong with the set up

Carbonate Samples; These numbers can be corrected offline in a spreadsheet, using the alpha for CO2(gas) vs CO2(carbonate). Alternatively, you can put the numbers 2. Computer:



a. Wake up the computer, i.e. move mouse or hit the “shift” key, log in if necessary (ID and password available from staff).

b. Access Isodat: i. Isodat’s Instrument Control and Isodat Acquisition programs should already be

running--with their buttons on the bottom button bar. ii. If there is a small red square on the right side of the bottom button bar, that means

Acquisition is acquiring data--the instrument is already in use--do not use it. iii. If Isodat is not running, open the Isodat 2.0 folder on the desktop and start

“Instrument Control” and then “Acquisition” by double clicking on the icons. These can also be found under the “start” button. Under instrument control, power up the mass spec’s source (click on the red sunburst on the menu bar in the “accessories” panel on the top left side of the Instrument Control window) and turn on the source heater (click on button to left of “source heater” in the MS State panel--on is “green”).

3. Mass Spec, XP: a. Analyzer and Inlet pumps: The analyzer pump and inlet pump buttons are on: i.e. it is pushed in and the red indicator light on that button is on. Get help if the analyzer pumps are off. b. Panel lights: NO red lights should be lit--only green ones, c. Panel lights, bottom section: The bottom two lights should be lit if the source is on. If they are off the can be turned on via the computer--Isodat Instrument Control, “Accessories” panel, click on red sunburst to turn on source, grey sun burst turns source off, and “lightning” icon toggles the high voltage on and off. Sometimes you may have to turn off the source then turn it back on to get it to work properly. 4. Check Instrument and Gas Configuration in Isodat: a. Configurations: in Isodat Acquisition, check the bottom left hand side of window for the following icons with the settings in the adjacent box: i. Toolbox icon: select “gasbench” if it is not already picked. ii. The gas icon (horn with bubbles coming out of it--hey, I didn’t design it): select “CO2”, it should have been in “standby” mode. b. Panel Settings: “MS” panel on left side of Isodat Acquisition window, top: High Voltage= ___ Magnet=___ HV= 3.04 (Not 0.3--click on “lightning” icon to turn HV back on if it’s at 0.3) Box ~0.5 Trap ~0.5 (may vary with current settings) Vac ~1.4 x 10-6 mBar Mass listed for N2: 28 29 30 The mV readings for the different masses may be high if the mass spec was recently opened to the Gasbench--or if there is a clog in the lines 5. Check Peripheral--Thermo Gasbench III and Gas tanks

a. Thermo GasbenchIII: b. PAL autosampler:

c. Check main He, Ref 1 and Ref 3 (CO2 in He, if being used) tanks: Gas tanks nest to the computer, tank pressures should be greater than 500 psi. 6. Set-up Run a. Open Sequence template in Isodat Acquisition (or Workspace) module, then immediately save it in your sequence folder as your current sequence with an appropriate name. b. Fill out sequence: ...



iv. Run isotope ratio standards (house reference material): run as “sample” types, in triplicate or quadruplicate like your samples. You should run at least two if not three different references, preferably bracketing the isotopic ratio of the samples, and one should be re-run atht end of a sequence to check for drift. Please note, there are only 49 slots in the autosampler, with ca. five used for junk, blanks and percent composition runs, leaving 44 available. With twelve (4x3) used for reference samples, thirty two are left for unknown samples. If you run in quadruplicate, that means 8 different samples can be run per autosampler load. v. If possible, group samples by isotopic ratio in a series and you need to run samples of the same type together. Depending on sample size and type, there can be memory effect. For a given series and size of samples, triplicate runs of known samples with differing isotopic ratios can be used to calculate the memory effect and then the corresponding correction applied to the data. Alternatively, discard the first run in the group and average the latter two only. vi. Fill out rest of sequence: peak center: check for all samples except last “standby” mode run. type: as discussed above--sample. blank or reference (amount% only) Identifier1: used in filename, only letters, numbers, dash and space allowed. preparation: additional information, e.g. gas pressure settings, sample prep. etc. method: should already be in your template, “PI name\method name” last run is “standby” method. other columns, e.g. “identifier2”, “comments”, available if needed c. Load Autosampler: That is, load sampling needle into holder. If needed. load in acid dosing needle too. Typically Gasbench protocols require equilibration in the sample holder at constant temperature for a day or more. Therefore the samples should be loaded already. 7. Start Run a. Double check gas pressures and GC temperature. Gasbench sample holder should have been at temperature during incubation phase. b. Wait for system to stabilize if you had to reset pressures/temperature. c. Double check options in Sequence. Click on the “Options” icon in the Sequence panel. This should be set up for you in the template. The folder name should be “\PI name\yourname\”. The “date” or “date and time” should be checked in the “post” selection to be appended to the listed name. If you type in a name following “...yourname\” , then the date or date and time will be appended to that name. The filename section should be blank--delete “Acquisition” if it appears”. The file name consists of the line number and identifier 1 in the “pre” section followed by the “data and time” in the post section. If you type a name in the box, it will fit in between those two sections. Printout and export options can also be adjusted at this time. If want to save the changes you’ve made, click on “ok” else “cancel”. d. Save Sequence, Start Run. Save sequence file, then click on green arrow icon in sequence panel to start run. If it was set up in “Workspace”, close sequence after saving it, go to “Aquistion”, open sequence, the start sequence as above (remember to delete the word “acquisition” if it appears in the filename window. When you hit “Start”, the “options” page opens up again, but this time “Start Sequence” appears in the top (blue) part of the window. If it’s ok, hit “ok”. Sometimes an error message may come up if you are in the wrong “mode”. In that case, pick “Gasbench” or “Gasbench-no acid”--it has to be the one that’s compatible with your method. 8. Monitor first sample, especially line up of sampling syringe, then check back when a your first real sample is running. Follow to first run to make sure it is running OK. Check back later to make sure standards ran OK and then watch your first sample or two to make sure they’re fine too--this is especially important for new types of samples. If something is wrong, e.g. standards are off or it turns out the sample sizes are too large (or small), click on the red “stop” button to abort the sequence. It will ask you if you want to finish current sample--answer “yes” if the sample has dropped already.



9. End of Run--shut down:

a. gas configuration should be in standby mode and conflo dilution on (last line of sequence should have done this) b. Remove sampling and acid dosing (if used) syringes from PAL holder and insert them into empty tubes for safe keeping c. Remove samples from Gasbench and clean tubes. d. Turn down (NOT off) gases--to ~10 to 15 psi. (ref 3, CO2 in He, should be off--valve shut via computer and tank itself closed off)

Using the Trace GC and GCC (combustion or pyrolysis) on the Mat253 1. Sample Preparation: A. Clean samples only pleased--and they should have been analyzed by GC (either FID or standard MS) already to verify that they are clean and have the species of interest in them. B. Sample size: Standard injection size is ~1 uL. You might be able to load more after working out injection conditions, but that will broaden your peaks and you’ll have to adjust the backflush timing to make sure no solvent peak gets through the furnace. The instrument can detect 50 nano-grams of a hydrocarbon (roughly 3.5 nano-moles carbon). The actual sensitivity for a given compound will depend on mode of injection, GC separation and peak broadening. C. Standard, conditioning and blank runs. Standard(s) can be co-injected with your sample--that is, the PAL autosampler can be programmed to pick up a small amount of a standard solution, say in position 98, then pick up your sample in any other position in the tray before injecting both into the GC. If the GC has not been used in a while, a few conditioning runs will be needed--mainly to reoxidize the oxidation column and moisten the Nafion tubing in combustion mode or recoat the pyrolysis reactor in pyrolysis mode. A blank or standard only run prior to your sample runs is advisable to verify column performance. 2. Computer: a. Wake up the computer, i.e. move mouse or hit the “shift” key. b. Access Isodat:

i. Isodat’s Instrument Control and Isodat Acquisition programs should already be running--with their buttons on the bottom button bar.


iii. If Isodat is not running, open the Isodat 2.0 folder on the desktop (or use Window’s Start menu) and start “Instrument Control” and then “Acquisition” by double clicking on the icons. Under instrument control, power up the mass spec’s source (click on the red sunburst on the menu bar in the “accessories” panel on the top left side of the Instrument Control window) and turn on the source heater (click on button to left of “source heater” in the MS State panel--on is “green”).

3. Mass Spec: a. Analyzer and Inlet pumps: The analyzer and inlet pump buttons are on: i.e. they are pushed in and the red indicator lights on the buttons are on. Get help if the any pumps are off. b. Panel lights: NO red lights should be lit--only green ones, and they all should be on. c. Panel lights, bottom section: The bottom two lights should be lit if the source is on. If they are off the can be turned on via the computer--Isodat Instrument Control, “Accessories” panel, click on red sunburst to turn on source, grey sun burst turns source off, and “lightning” icon toggles



the high voltage on and off. Sometimes you may have to turn off the source then turn it back on to get it to work properly. 4. Check Instrument and Gas Configuration in Isodat: a. Configurations: in Isodat Acquisition, check the bottom left hand side of window for the following icons with the settings in the adjacent box: i. Toolbox icon: select “TraceGC” if it is not already picked. ii. The gas icon (horn with bubbles coming out of it--hey, I didn’t design it): select “CO2” for combustion mode, “H2” for pyrolysis mode, it should be in “standby” mode b. Panel Settings:

i. “MS” panel on left side of Isodat Acquisition window, top (CO2 mode): High Voltage= 9.005 Magnet=5512 HV= 9 (Not 0.3--click on “lightning” icon to turn HV back on if it’s at 0.3) Box ~0.2 Trap ~0.8 Vac ~3.4 x 10-6 mBar Mass listed for CO2: 44 45 46 The mV readings for the different masses should be around 2-3 mV. ii. The MS State panel should have the “pumps on”, “src pump...”, “src vacuum...”, “set point src”, and “src heater” buttons green (on), other heaters should be off (grayed out). iii. Focus 253: All the bars should be between 1/4 to 3/4 of full scale. The emission should be at 1 mA (if it’s lower, make sure it is not in standby mode). The electron energy should be ~ 76 V. Occasionally, it bottoms out (minimum setting 50 V)--get help. 5. Check Peripheral--Trace GC, GCC and Gas Tanks. A. Trace GC: The GC should be in standby mode with the standby light on (amber). It may also be in “gas saver” mode. If a run just finished, it may be in ready mode (green light) if it’s set to “auto-prep” run. To see the condition of the oven or inlets, press “oven”, or “right inlet”, or “right carrier” keys to see current state. Currently we’re only using the right injector, the PTV, so the left inlet and carrier should be off. The oven and right inlet should be between 40-60 oC, depending on last run, and the carrier should be set to 2 mL/min--though this is sample/method dependant. Please note, these are software adjustable--with a gc method programmed in Isodat 2.0. B. i. GCC: combustion mode. Oxidation oven should be set to 940 and reduction oven to 650. When set up in combustion mode, the temperature of the ovens is maintained. Pyrolysis oven (controller beneath GC) is off, that is the readings are roughly room temp. Oxygen pressure on GCC in 0.5 bar, ref 1 should be hooked up to the CO2 tank and settings can be 10 to 35 psi depending on run, and He is usually set to 15 to 20 psi. Please note your settings for future use--put them in the comment section of log book. If the GCC is just being set up for combustion work, make sure GC column and outlet line are connected to the upper back furnace (combustion furnace). In the GCC, the outlet line from the second valco valve runs to the Reduction furnace that is connected to the drying tube which is connected to the open sample split. ii. GCC: pyrolysis mode: Pyrolysis (TC) furnace should be at 1400 (controller underneath GC), GCC Oxidation and Reduction furnaces should roughly be at room temperature. Oxygen is not used, but usually kept at pressure, and H2 should be connected to ref 1. The H3+ factor needs to be run every day



Helpful Hints from .... aka, keeping things running.... Conflo: 1. Dilution doesn’t dilute/triggers apparent air leak.

a. He capillary that goes to sample open split is sticking when it’s being lowered. Close off mass spec, remove all capillaries from sample open split and clean them and the test tube that is the open split. Use ethanol on kimwipes, make sure you sponge up all the liquid from the test tube. Clip off small end pieces from the capillary, and re-insert all into test tube, biggest to smallest. Let He blow through to dry out tube and capillaries for a few hours before opening up to mass spec.

b. Clogged/loosened vent lines on sample switching valve in Conflo. GCC: 1.

Documents

Using the Mass Specs--general instructions 1 2 3 4earth.yale.edu/sites/default/files/files/ESCSIS/ESCSIS-man_prelim.pdf · HV= 3.04 (Not 0.3--click on “lightning” icon to turn