TITLE: Dissolved inorganic carbon concentration and 13C/12C
AUTHORS: Daniel Nothaft

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>CO</span><span style = "vertical-align:sub;">2</span><span> conversion by H</span><span style = "vertical-align:sub;">3</span><span>PO</span><span style = "vertical-align:sub;">4</span><span>, extracted CO</span><span style = "vertical-align:sub;">2</span><span> introduced into continous flow IRMS for </span><span style = "vertical-align:super;">13</span><span>C/</span><span style = "vertical-align:super;">12</span><span>C analysis</span></div><div class = "text-block"><span>This method follows the general principles described in Assayag et al. (2006), which emphasizes the importance of maintaining the same water : H</span><span style = "vertical-align:sub;">3</span><span>PO</span><span style = "vertical-align:sub;">4</span><span> volume ratio for standard solutions and samples (ratio of 10 in this case) in order to obviate issues regarding isotopic fractionation between CO</span><span style = "vertical-align:sub;">2</span><span> (g) and CO</span><span style = "vertical-align:sub;">2</span><span> (aq).</span></div><div class = "text-block">References</div><div class = "text-block">     Assayag, N., Rivé, K., Ader, M., Jézéquel, D., and Agrinier, P. (2006). Improved method for isotopic and quantitative analysis of dissolved inorganic carbon in natural water samples. Rapid Communications in Mass Spectrometry 20, 2243–2251 
 
  
 
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[STEPS]
?. [Laboratory preparation]
Plan samplesBefore prepping vials, you must consider what range of dissolved inorganic carbon (DIC) you expect in your samples.The sensitivity range is determined by the method blank, the water volume, the lower limit of mass for which we can consistently measure carbonate standards (~10 µg), and the detector saturation / linearity effects on the isotope ratio mass spectrometer (IRMS). DBN has optimized procedures to lower blanks, which are reflected in this procedure, although further blank reduction is an ongoing project.Below are some guidelines of appropriate volumes to use:A 1 mL water sample in a 12 mL Exetainer will be good for most freshwater and marine systems (sensitivity range ~ 300 – 4000 µM on Thermo GasBench - Delta V IRMS).If you expect [DIC] > 4000 µM, you can use less sample (i.e. 0.5 ml water). If you expect [DIC] If your system has a wide range of [DIC], you can take replicate samples at different water volumes (i.e. for Oman ophiolite, where [DIC] ranges ~3500 -  Plan method blanksAlso, consider what you will use as a method blank. At minimum, you must purge (1-3) vial(s) with helium during sample preparation, and subsequently add decarbonated water + H3PO4.If you plan to have a quick turnaround time between preparing samples and analyzing them (If the period between sample preparation and analysis is longer (weeks to months), or if you have any doubt that you will return with samples (due to mishaps in field or new experimental design etc.), you should probably wait to prepare standards until a day before analysis to ensure that your standards are unaltered and to avoid wasting time and standards for a run that never occurs.If the latter applies to you, then prepare a set of blanks as you prepare your samples and another set of blanks the day prior to analysis to check for 1) diffusion between sample preparation and analysis and 2) the repeatability of your blank preparation. Ideally, blanks should be brought into the field because diffusion is a function of pressure gradients and temperature, which may be different in the lab compared to your sampling site. In addition to the He purge + water blank mentioned previously, He-only, He + H3PO4 only, and/or He + carbonate mineral standard + water + H3PO4 can be prepared to test for CO2 loss/gain, and to determine the sources error, if necessary.The steps below describe the full laboratory procedure for preparing samples and/or standards. Depending on your specific sampling and blank preparation strategy, some steps may not apply. In any case, stop when you reach the centrifugation step, and finish centrifugation and equilibration the day prior to analysis on the IRMS.
For 2018 Oman samples, DBN left blanks in the lab; in 2019,  DBN brought blanks to the field.DBN has not noticed significant change in blanks over ~1 month time scales.
?. Combust with aluminum foil covering the mouth of the Exetainer in muffle furnace 450 ˚C for 5 hr.
DBN has found that Exetainers (with septum installed) are consistently 11.70±0.01 (1s) mL. This is sufficiently consistent that the volume of Exetainers do not need to be individually measured before sampling for DIC concentration. Since the Exetainers are continually exposed to acid, it is not necessary to give them a separate acid wash.
?. [Laboratory preparation]
Measure volume of vialsDBN has found that the nominally 100 mL vials (with septum installed) actually have a mean volume of 117 mL and can vary by a few mL.This may be sufficiently consistent for some purposes, and DBN has not attempted to correct for these differences so far, but moving forward, DBN recommends measuring the volumes of all 100 mL vials since they may have come from various sources over the years, so it is nice to know the volumes, and correct for differences between vials, if necessary.To measure volume:Label vials with unique identifiers (in Sharpie permanent marker)Record lab temperature (pH probe in Templeton lab has thermometer)Cap vial with blue butyl stopperMeasure mass of vialUsing 2 needles and a 60ml syringe, fill vial all the way with deionized water. Make sure there are no bubbles.Re-measure mass of vialCalculate volume of water contained in vial https://www.engineeringtoolbox.com/water-density-specific-weight-d_595.html
It is best to measure all vials at the same time to avoid any systematic offsets between populations of vials.
DBN is planning to measure the volume of a bunch of 100 mL vials, and then keep a stock of them just for this purpose, to avoid repeatedly spending time measuring vial volumes.
?. Weigh out carbonate standards on micro balance, and label standard vials.
?. [Laboratory preparation]
Acid washSince 100 mL serum vials are used for a variety of lab tasks, soak them in an acid bath overnight to remove any  carbonate precipitates that may be in the vials.
?. [Laboratory preparation]
Combust with aluminum foil covering the mouth of the vial in muffle furnace 450 ˚C for 5 hr.
If you have calibrated the vial volumes, write the vial ID # on the aluminum foil in Sharpie marker with enough force to create an imprint in the foil. The sharpie will burn off, but the imprint will remain. Re-label after the vials have cooled down.
?. Cap vials Gently screw the cap onto the vial until slight resistance is felt with the septum. Then turn the cap an additional one quarter turn. Looking at the top of the cap on the sealed vial from above, a very slight indentation of the septum may be seen through the aperture of the cap, indicating a good seal If the cap has a puckered look, it has been overtightened. If this is the case, replace with new cap. http://www.labco.co.uk/information/user-guide
?. Boil waterUse acid washed glassware if possible, but ultra-pure (MilliQ) water rinsed is OK if you don’t have acid-washed glassware prepped.Rinse an Erlenmeyer flask for the H3PO4 and a beaker for the water with ultra-pure H2O from Kopf lab, BESC 335. Rinse 2 small stir bars (Cover beaker/flask with aluminum foil but leave a little room for venting. This helps it heat up faster and avoids contamination. Put the water on the small VWR hotplate in the hood in rm. 345, heat setting 10, stir setting 1. Depending on volume of water, this may take ~15 min to get boiling. Wait 10 min from first observation of bubbles. Then turn off heat, wait for liquid to stop boiling, and then remove from hot plate.Rinse 2 small stir bars (Cover beaker/flask with aluminum foil but leave a little room for venting. This helps it heat up faster and avoids contamination. Put the water on the small VWR hotplate in the hood in rm. 345, heat setting 10, stir setting 1. Depending on volume of water, this may take ~15 min to get boiling. Wait 10 min from first observation of bubbles. Then turn off heat, wait for liquid to stop boiling, and then remove from hot plate.
Make sure you select an Erlenmeyer flask that is an appropriate size such that the syringe you are using for H3PO4 transfer can fit in and reach near the bottom of the flask. Otherwise, you will end up wasting H3PO4. You may need to prepare multiple batches of H3PO4 depending on the number of samples you have.
Ultra-pure water can overheat and then violently boil since it lacks impurities which are good nucleation points for boiling. DBN has not had any issues with this happening, but would suggest using older glassware and stirbars that are more likely to have tiny nicks and scratches, which can act as nucleation points for boiling. Alternatively (and probably optimally, although DBN has not used yet for this method), consider adding boiling stones to water and/or H3PO4.
?. Purge vials with HeliumHe purge the vials for 5 minutes Put the purging needles in the septa such that the inflow needle is a bit deeper than the outflow needle. This allows you to pull both needles out at the same time with the outflow needle being extracted first. It is best to avoid wildly different partial pressures of He between vials, so it is good to do this consistently.It is good to have needle outflow connected to tube that is submerged in water. this prevents air leakage when removing needles.Approximately 5 bubbles per second should emanate from the outflow tubes submerged in water.
?. [Field sampling]
Take to the field He-purged Exetainers as described in Section 'Laboratory preparation before sampling.'
?. [Field sampling]
Taking samplesFill a 3 mL syringe with sample water and attach 0.2 µm polycarbonate in line filter and needle.Discard water.Refill the syringe with sample water. Ensure there are no bubbles. Attach filter and needle. Discard water until only the sample volume is left in syringe.Inject water into exetainer in same manner as standards (right-side up).Withdraw syringe swiftly, holding by the needle so it doesn't come loose in the septum.
If water is being pumped from subsurface and there is concern about the sample equilibrating with atmosphere, then it is probably best to sample directly from the pump stream (i.e. through a luer-lock port on a manifold), but if ease of sampling is an issue and/or the water being sampled is naturally open to air (i.e. sampling a river or sampling from a boat), sampling is easier and more reproducible if a sub-sample reservoir is taken (i.e. a 10L bucket, washed out three times with site water.)
Consider taking samples in duplicate or triplicate if possible.
Be sure to keep needles and filters sterile between sample replicates.
Some people like to store samples upside down which allegedly decreases gas leakage across the septa, but I am not aware that anyone has tested this. I would say it does not matter as long as you are consistent with sample storage in all samples. I would recommend bringing a tube rack to field to stabilize samples during transport. If you do store samples upside down, make sure to do the centrifugation step during sample prep.For Oman samples, DBN stored samples rightside in 2018 and upside-down in 2019.
?. Prepare H3PO4Using stand with ring attachment and glass funnel (preferably acid-washed) in the hood, carefully transfer 85% H3PO4 to the Erlenmeyer flask. Be careful not to fill it up all the way (3PO4 expands and bubbles upon heating.Boil according to the same steps as for water.Transfer to vials in the same manner as for water, but do not use a syringe filter.
Use safety goggles, lab coat, and nitrile gloves while handling H3PO4.
Use water:H3PO4 ratio of 10, i.e. if using 1 mL water sample, use 100 µL 85% H3PO4.DBN has confirmed that this reduces pH of hyperalkaline Oman water from >11 to
As it turns out, hot acid is not the best thing for plastic syringes. If you notice that the luer-lock threads are not connecting well, start using a new syringe to avoid a needle coming loose in the vial.
?. Clean off vialsAfter injections are finished, carefully wipe the tops of the Exetainer with paper towel / kimwipe so that no fluid remains on the septa.
?. CentrifugationI recommend centrifugation, which serves two purposes: (1) getting liquid off the top of the septum, which can potentially enter the GasBench needle and get into the capillary and (2) ensuring that no CaCO3 powder sticks to the sides of the Exetainer, which can throw off your calibration.Before centrifuging standards, visually inspect if there are any bits of carbonate stuck to the sides of the exetainer. If there are, try to swoosh the liquid around to scoop it up.Centrifuge all samples 5 minutes at 2500 rpm on Templeton lab centrifuge.After centrifuging, visually inspect the standard solutions again for sticking particles. If necessary, repeat swooshing liquid and centrifuging until there are no more sticking particles.
Mass must be balanced on either side of centrifuge. Add another vial to balance centrifuge if necessary.
?. EquilibrationAfter centrifuging, place exetainers in tube rack on shaker table (upright, with caps on top). Shake at ~150 RPM for 15-18hr. Ideally, do this in the stable isotope lab so it is at same temperature as it will be analyzed at.
?. Ensure that heat block on GasBench is turned off well before samples are analyzed (a few hours to overnight).
?. Centrifuge againThis may sound excessive, but I would recommend centrifuging again right before analysis. The reason is that condensation often forms while the samples are shaken overnight. This especially true for standards that had hot water added to them the previous day. Centrifuging again probably saves time overall by reducing the likelihood of GasBench needle clogging.
?. [Laboratory preparation]
Weigh out carbonate standards on micro balance, and label standard vials.
?. [Laboratory preparation]
Cap vialsInsert thick blue chlorobutyl stopper.Secure aluminum crimp top with crimper.
The blue stoppers are pretty thick and tough to get in the vial. DBN usually autoclaves them before inserting into vial, which makes them more pliable and slick.
In 2018, DBN used a thinner gray chlorobutyl stopper with PTFE lining, but the blue chlorobutyl stopper mentioned here is thicker and gives better seal, so the blue one is recommended.
?. [Laboratory preparation]
Purge vials with HeliumHe purge the vials for 30 minutesPut the purging needles (23g - 0.064cm x 2.5 cm) in the stopper such that the inflow needle is a bit deeper than the outflow needle. This allows you to pull both needles out at the same time with the outflow needle being extracted first.It is best to avoid wildly different partial pressures of He between vials, so it is good to do this consistently.It is good to have needle outflow connected to tube that is submerged in water. this prevents air leakage when removing needles. Approximately 5 bubbles per second should emanate from the outflow tubes submerged in water.Use new needles for each puncture of the stopper.
?. [Laboratory preparation]
Boil waterUse acid washed glassware if possible, but ultra-pure (MilliQ) water rinsed is OK if you don’t have acid-washed glassware prepped.Rinse an Erlenmeyer flask for the H3PO4 and a beaker for the water with ultra-pure H2O from Kopf lab, BESC 335. Rinse 2 small stir bars (Cover beaker/flask with aluminum foil but leave a little room for venting. This helps it heat up faster and avoids contamination. Put the water on the small VWR hotplate in the hood in rm. 345, heat setting 10, stir setting 1. Depending on volume of water, this may take ~15 min to get boiling. Wait 10 min from first observation of bubbles. Then turn off heat, wait for liquid to stop boiling, and then remove from hot plate.Rinse 2 small stir bars (Cover beaker/flask with aluminum foil but leave a little room for venting. This helps it heat up faster and avoids contamination. Put the water on the small VWR hotplate in the hood in rm. 345, heat setting 10, stir setting 1. Depending on volume of water, this may take ~15 min to get boiling. Wait 10 min from first observation of bubbles. Then turn off heat, wait for liquid to stop boiling, and then remove from hot plate.
Make sure you select an Erlenmeyer flask that is an appropriate size such that the syringe you are using for H3PO4 transfer can fit in and reach near the bottom of the flask. Otherwise, you will end up wasting H3PO4. You may need to prepare multiple batches of H3PO4 depending on the number of samples you have.
Ultra-pure water can overheat and then violently boil since it lacks impurities which are good nucleation points for boiling. DBN has not had any issues with this happening, but would suggest using older glassware and stirbars that are more likely to have tiny nicks and scratches, which can act as nucleation points for boiling. Alternatively (and probably optimally, although DBN has not used yet for this method), consider adding boiling stones to water and/or H3PO4.
?. [Laboratory preparation]
Prepare H3PO4Using stand with ring attachment and glass funnel (preferably acid-washed) in the hood, carefully transfer 85% H3PO4 to the Erlenmeyer flask. Be careful not to fill it up all the way (3PO4 expands and bubbles upon heating.Boil according to the same steps as for water.Transfer to vials in the same manner as for water, but do not use a syringe filter.
Use safety goggles, lab coat, and nitrile gloves while handling H3PO4.
Use water:H3PO4 ratio of 10, i.e. if using 100 mL water sample, use 50 mL 85% H3PO4.DBN has confirmed that this reduces pH of hyperalkaline
As it turns out, hot acid is not the best thing for plastic syringes. If you notice that the luer-lock threads are not connecting well, start using a new syringe to avoid a needle coming loose in the vial.
?. [Laboratory preparation]
Clean off vialsAfter injections are finished, carefully wipe the tops of the Exetainer with paper towel / kimwipe so that no fluid remains on the septa.
?. [Laboratory preparation]
CentrifugationI recommend centrifugation, which serves two purposes: (1) getting liquid off the top of the septum, which can potentially enter the GasBench needle and get into the capillary and (2) ensuring that no CaCO3 powder sticks to the sides of the vial, which can throw off your calibration.Before centrifuging standards, visually inspect if there are any bits of carbonate stuck to the sides of the exetainer. If there are, try to swoosh the liquid around to scoop it up.Centrifuge all samples 5 minutes at 2500 rpm on Templeton lab centrifuge.After centrifuging, visually inspect the standard solutions again for sticking particles. If necessary, repeat swooshing liquid and centrifuging until there are no more sticking particles.
Mass must be balanced on either side of centrifuge. Add another vial to balance centrifuge if necessary.
?. [Laboratory preparation]
EquilibrationAfter centrifuging, place exetainers in tube rack on shaker table (upright, with caps on top). Shake at ~150 RPM for 15-18hr. Ideally, do this in the stable isotope lab so it is at same temperature as it will be analyzed at.
?. [Laboratory preparation]
Ensure that heat block on GasBench is turned off well before samples are analyzed (a few hours to overnight).
?. [Field sampling]
Taking samplesFill a syringe with sample water and attach 0.2 µm polycarbonate in line filter and needle.Discard water.Refill the syringe with sample water. Ensure there are no bubbles. Attach filter and needle. Discard water until only the sample volume is left in syringe.Inject water into exetainer in same manner as standards (right-side up). Withdraw syringe swiftly.
If water is being pumped from subsurface and there is concern about the sample equilibrating with atmosphere, then it is probably best to sample directly from the pump stream (i.e. through the luer-lock port on manifold), but if ease of sampling is an issue and/or the water being sample is naturally open to air (i.e. sampling a river or sampling from a boat), sampling is easier and more reproducible if a sub-sample reservoir is taken (i.e. a 10L bucket, washed out three times with site water.)
Consider taking samples in duplicate or triplicate if possible.
Be sure to keep needles and filters sterile between sample replicates.
Some people like to store samples upside down which allegedly decreases gas leakage across the septa, but I am not aware that anyone has tested this. I would say it does not matter as long as you are consistent with sample storage in all samples. I would recommend bringing a tube rack to field to stabilize samples during transport. If you do store samples upside down, make sure to do the centrifugation step during sample prep.For Oman samples, DBN stored samples rightside in 2018 and upside-down in 2019.
?. [Field sampling]
Take to the field He-purged Exetainers as described in Section 'Laboratory preparation.'
?. [Laboratory preparation]
Centrifuge againThis may sound excessive, but I would recommend centrifuging again right before analysis. The reason is that condensation often forms on the underside of the stopper while the samples are shaken overnight. This is especially true for standards that had hot water added to them the previous day. Centrifuging again probably saves time overall by reducing the likelihood of GasBench needle clogging.
?. [Running samples on GasBench - Delta V IRMS]
Check He tank pressure and write down in Delta notebook
?. [Running samples on GasBench - Delta V IRMS]
Go to Isodat acquisition program.
?. [Running samples on GasBench - Delta V IRMS]
Check in bottom left that it is on ConFlo IV + gasbench & CO2 mode
?. [Running samples on GasBench - Delta V IRMS]
Turn on reference gas dilutions to make a Voltage in range of what you expect for your samplesConFlo IV Diagnosis > Reference DilutionEngage reference dilutions 1 and 2Write down the dilution setting in Delta lab notebook
?. [Running samples on GasBench - Delta V IRMS]
Turn on Reference gas
It’s a good idea to turn on reference gas right away b/c it helps flush any water vapor out of the system
?. [Running samples on GasBench - Delta V IRMS]
Write down HV, Vac, Box, Trap
?. [Running samples on GasBench - Delta V IRMS]
Run autofocusIcon with 2 blue bars in upper leftWrite down mass 44 voltage in delta lab book
?. [Running samples on GasBench - Delta V IRMS]
Pass to gas configuration
?. [Running samples on GasBench - Delta V IRMS]
Write “pass to gas config” next to mass 44 voltage in delta lab book
?. [Running samples on GasBench - Delta V IRMS]
Turn off reference CO2 tankGive mass spec ~1 min to let this gas flush out.Write down the background voltages for Mass 44, 45, and 46
?. [Running samples on GasBench - Delta V IRMS]
Switch to ConFlo IV mode
?. [Running samples on GasBench - Delta V IRMS]
Run On/Offs & linearityOpen -> Thermo -> Isodat NT -> Global-> User -> Conflo IV Interface -> Conflo IV Interface -> Sequence-> 20171024 CO2 On Off Lin.seqRun ‘CO2 On Off.met’ and ‘CO2 Linearity.met’Say OK to all the run settingsThe point of this is basically to determine ifthe mass spec is running properlyFor the On/Offs, 𝛿18O should have a standard deviation of ~0.01 per mil Linearity results with standard deviation 18O is fine
?. [Running samples on GasBench - Delta V IRMS]
Load Exetainers in GasBench autosampler
?. [Running samples on GasBench - Delta V IRMS]
Set up sequence
?. [Running samples on GasBench - Delta V IRMS]
Select all fields in your sequence and start data acquisition.
?. [Running samples on GasBench - Delta V IRMS]
Check He tank pressure and write down in Delta notebook
?. [Running samples on GasBench - Delta V IRMS]
Go to Isodat acquisition program.
?. [Running samples on GasBench - Delta V IRMS]
Check in bottom left that it is on ConFlo IV + gasbench & CO2 mode
?. [Running samples on GasBench - Delta V IRMS]
Turn on reference gas dilutions to make a Voltage in range of what you expect for your samplesConFlo IV Diagnosis > Reference DilutionEngage reference dilutions 1 and 2Write down the dilution setting in Delta lab notebook
?. [Running samples on GasBench - Delta V IRMS]
Turn on Reference gas
It’s a good idea to turn on reference gas right away b/c it helps flush any water vapor out of the system
?. [Running samples on GasBench - Delta V IRMS]
Write down HV, Vac, Box, Trap
?. [Running samples on GasBench - Delta V IRMS]
Run autofocusIcon with 2 blue bars in upper leftWrite down mass 44 voltage in delta lab book
?. [Running samples on GasBench - Delta V IRMS]
Pass to gas configuration
?. [Running samples on GasBench - Delta V IRMS]
Write “pass to gas config” next to mass 44 voltage in delta lab book
?. [Running samples on GasBench - Delta V IRMS]
Turn off reference CO2 tankGive mass spec ~1 min to let this gas flush out.Write down the background voltages for Mass 44,45, and 46
?. [Running samples on GasBench - Delta V IRMS]
Switch to ConFlo IV mode
?. [Running samples on GasBench - Delta V IRMS]
Run On/Offs & linearityOpen -> Thermo -> Isodat NT -> Global-> User -> Conflo IV Interface -> Conflo IV Interface -> Sequence-> 20171024 CO2 On Off Lin.seqRun ‘CO2 On Off.met’ and ‘CO2 Linearity.met’Say OK to all the run settingsThe point of this is basically to determine ifthe mass spec is running properlyFor the On/Offs, 𝛿18O should have a standard deviation of ~0.01 per mil Linearity results with standard deviation 18O is fine
?. [Running samples on GasBench - Delta V IRMS]
Manual injection on gasbenchMake sure autosampler is disabled in Isodat acquisitionStart acquisitionGo to GasBenchHold GasBench needle firmlyStab straight down into the vialMake sure both holes of GasBench needle are below stopper.
?. Inject water in standard vials
Boiling water helps reduce CO2 background, but if water or acid is too hot, it will deform the syringe tip, which can result in your needle getting stuck in the vial, which is no good.
Use 'nurse technique' to remove bubbles from syringe (invert syringe and flick while pushing out liquid. waste container can be useful for this.)
If you are using a syringe filter for field samples (strongly recommended), I would recommend also using one in water injections of standards. If doing this, hold on to the needle as you pull it out of the Exetainer to avoid the needle falling off and degassing occurring.
If you have a lot of samples, and water injections are taking long enough that your water is cooling down, you may consider putting it back on the hotplate at low heat (~heat setting 2) to keep warm.
For water and acid additions in Exetainers, it is OK to re-use needles between vials.
?. [Laboratory preparation]
Inject water in standard vials
Boiling water helps reduce CO2 background, but if water or acid is too hot, it will deform the syringe tip, which can result in your needle getting stuck in the vial, which is no good.
Use 'nurse technique' to remove bubbles from syringe (invert syringe and flick while pushing out liquid. waste container can be useful for this.)
If you are using a syringe filter for field samples (strongly recommended), I would recommend also using one in water injections of standards. If doing this, hold on to the needle as you pull it out of the vial to avoid the needle falling off and degassing occurring.
If you have a lot of samples, and water injections are taking long enough that your water is cooling down, you may consider putting it back on the hotplate at low heat (~heat setting 2) to keep warm.
For water and acid additions in with the blue butyl stoppers, use new needles each time.