Water Extractions (Cryogenic Water Distillation) For H and O stable isotope analyses




To analyze the isotope composition of the water contained in leaf, stem, or soil samples, we use the water extraction method to completely remove the water from the sample without isotopic fractionation. This method both draws and drives the water from your sample to the collection vessel by applying vacuum pressure to the extraction unit and extreme differences in temperature between the sample vessel and the collection vessel. As you heat your sample under vacuum, the water will leave your sample and condense in the frozen collection vessel. It is critical to allow each sample enough time to completely dehydrate so that no fractionation will occur and we suggest the following extraction times for different types of samples following the tested extraction times of West et al. (2006):

Recommended extraction times for different materials:
Soils: 40-45 min
Stems: 60-70 min
Leaves: ~60 min

Be aware that not all samples are behave equally and therefore water extraction times and the ease with witch the method is used varies. Soil type, leaf and stem morphology, and the water content of all samples vary widely so you may need to adjust these suggested extraction times based on your own sample’s properties. It is never a problem to run a sample for too long, but removing a sample from the extraction line prematurely will destroy the sample.

Run a method-check standard (Recommended: Brooks Well Water or BWW, {SUP()}2{SUP}H= -37.2 ∓ 1.0 and {SUP()}18{SUP}O= -5.27 ∓ 0.08) through the extraction process each time you use the extraction line to verify your technique and treat this standard water in the same way you handle your samples. This means that you should extract this 200μl of water for the same time interval as your samples and if you add anything to your sample vessel to filter particulates (cotton-ball or glass wool for soil samples) you should add it to you standard extraction as well. We record these data to monitor the long-term error associated with the extraction method. As a word of warning, this method is prone to operator error and samples are lost during extraction due to leaks and loss of vacuum during the extraction. To prevent sample loss, do not operate more stations than you feel comfortable running alone and collect duplicate samples if possible for extraction in case the original sample is compromised during extraction.

PLEASE remember to wear safety glasses at all times (NO EXCEPTIONS) and gloves when working with liquid nitrogen and the boiling water.


Preparing the Stations for Your Samples
If you have done extractions before and you just need a refresher, you can read the most important instructions in __bold__.

===__NOTE:__=== For those experienced with the water extraction protocol and aware of the status of extraction lines, you can skip the “zero-point-o-the-day” check before starting samples (skip steps 3-8). Do this ===ONLY=== if you observe a clean layer of grease around all of the exposed ground-glass joints (and therefore expect a good seal) and you know that previous users of the line have maintained the cleanliness of the ultra-torr connectors. If you skip the cleaning and put a sample on the line at the beginning of the day, you will check for leaks after you pull a vacuum on your station while the sample is frozen. ”__Keep the sample frozen until you verify that the station is not leaking.__” If the station does not leak, record this vacuum pressure as the “zero-point-o-the-day” value and proceed with the method. If it does leak, immediately remove your sample and keep it frozen while you then clean the station. (This shortcut may provide a more reliable assessment of station -leakiness’ because the vacuum gauge is sensitive to changes in temperature and this tests for leaks at a constant freezing temperature.)

Each station must be clean and able to pull and maintain a vacuum before you sacrifice any samples. Follow these instructions:

#__Fill up the water trap with liquid nitrogen.__ This will lower the temperature and increase vacuum pulled by the pump. Additionally, this will trap any water vapors in the line from going into the pump.%%% %%%
#__Make sure that the manifold (main line) vacuum gauge reads between 0-10 millitorr (mT) (0 is best).__ [[If all the stations are closed, and the manifold gauge does not zero there are two things you can do. First, make sure that the gauge is reading properly, and the connecting junction is open. Second, check the connection between the main line and the water trap. If it looks dirty or has air bubbles in it, it may need to be cleaned and re-greased.]%%% %%%::__(Steps for room-temperature “zero-point-of-the-day” and station cleaning)__::
#Test each station independently, and __make sure that each station is able to “zero”.__ The gauge for each station generally stops between 10-50 mT (due to offsets in the gauge), and if this is the actual zero point, the manifold gauge will return to its zero level again. It is useful to write down the zero-point-o-the-day on a piece of lab tape and stick in on the vacuum gauge for each station. This will be helpful later when you are running samples.%%% %%%
#If a station does not zero, then you will need to clean and re-grease all the pieces (see ~np~#~/np~5). If the station zeros well, skip to step ~np~#~/np~7.%%% %%%
#Pieces that need cleaning/ light re-greasing:
+__a.__ Ball valve near top of station. Smooth greasing on both parts of valve- no clumps of grease. Junction should appear clear and clean.
+__b.__ Ultra-torr connections. There are 2 large Ultra-torrs that hold the sample vial and the collection vial onto the station (2 connections each), and 1 small Ultra-torr (1 connection) that attaches to the vacuum gauge. Take apart each Ultra-torr connection, and remove O-ring. Clean everything with kimwipes and ethanol (including the threads on the Ultra-torrs and all the O-rings). Re-grease O-ring, making sure that there are NO CLUMPS or grease, just a smooth, shiny surface. Return Ultra-torrs to their original position, and screw them on finger tight.
+__c.__ Sample vial junction.
+”i.” If you are using the exetainer (screwcap) set up, you will need to remove the O-ring and cap from the rod. Clean off rod, cap, and O-ring with a kimwipe (and ethanol if you want). Lightly grease the cap threads, the O-ring, and the threads on the blank vial you will use for zeroing. Put the cap back on the rod, and fit the O-ring snuggly against the lip of the rod. It must lay flat or else the vial will not screw on tightly. Screw on vial, and make sure that the vial is sitting straight and does not easily pull off. If vial is crooked or can be pulled off easily, use a new cap and start over. Also make sure there are no bulges in the O-ring that will cause a leak.
+”ii.” If you are using the sample vessel method, you will need to check the O-ring. If there is too much grease, too little grease, or any hair, dust, or glass wool particles, then there will be a leak at this junction. Clean off the O-ring, and both grooves of the sample vessel (upper and lower). Grease O-ring (light coat to make it shiny), and check carefully for any particles. If it looks clean, then replace it in the sample vessel groove, and clamp together.%%% %%%
#If you regrease everything and the station still won’t zero, do it all again. Be extra careful that there are no specks of dust or small hairs on the O-rings. If problems persist, there may be cracks in the glass or problems with the manifold. If needed, change parts or skip that station.%%% %%%
#Once you do get your station to zero, you want to __test the station make sure there are no leaks__. To test for leaks, close off the station once it’s “zeroed”, and note the mT reading (make sure you are looking at the right station number!). Watch the pressure gauge for 1 minute. A leakage rate of up to 30 mT per minute is acceptable, but the less it leaks the better.%%% %%%
#When your stations all zero and have very low leakage rates, then you are ready to go!

===__Setting Up for Water Extractions__===
#Make sure there is a minimum of 10 liters of liquid nitrogen (LN{SUB()}2{SUB}) in the large tank. Fill your 5L dewer with LN{SUB()}2{SUB} (wear safety glasses and gloves).%%% %%%
#Start boiling some hot water on the large heat plate. Also, fill up the small (250ml) beakers that you will need, and heat them up on their respective hot plates with boiling chips to control for splatter.%%% %%%
#Get your samples organized and ready to go. Leave samples frozen at -20C if you will not extract them immediately.%%% %%%
#Fill up 1 short dewer (with blue mesh on the outside) and n tall silver dewers with LN{SUB()}2{SUB} (”n” = number of stations you are using). Fill the short dewer up to the top, and the tall dewers about half way.

===__Preparing the Sample: Exetainer sample vial__===
*Many people overfill the exetainer vials when they are collecting samples, which can clog up the water extraction system. If the vials are too full, then you can remove some of the sample from the vial and run it in the sample vessel (no screw cap), and save the remainder of the sample in the exetainer.

Is my sample vials too full Yes if:
a.) It is packed with stems and or leaves.
Only run half of the sample, and cut it up.

b.) There is a lot of soil in the vial and it looks moist (if you can see condensation or crystals in the vial, it’s moist!).
Only run half of the sample.

(See Figure below)
{img fileId=”146″ thumb=”y” alt=”” rel=”box[g]”}

===__Running your Samples: Exetainer sample vials or Sample Vessels__===
#__Make sure the valve of the station is closed before you remove any tubes.__ Twist the station knob finger tight until you see the O-ring enlarge so that it is plugging the line.%%% %%%
#Loosen the Ultra-torr and __remove the sample vessel from the station__. If you are using the exetainer vials set up, unscrew the blank vial (from the zeroing) from the cap, and put it away in the drawer. If you are using the sample vessel, unscrew the connecting clamp and separate the vessel and the glass cap.%%% %%%
#Choose the sample you will use, and __write down the sample name and station number in the EXTRATION BINDER.__%%% %%%
#Prepare a strip of kimwipe and get forceps ready. For exetainer vials, unscrew your sample vial, and wipe the inside of the cap with the strip of kimwipe. Then put this kimwipe piece into your sample vial, and push your sample down so that at least the top 1/3 of the vial is empty. For the sample vessel, open your sample vial, and fill up 1/4 to 1/3 of the sample vessel. Use a strip of kimwipe to clean out any condensation from your original sample vial, and put the kimwipe in the sample vessel with your sample.%%%~np~*~/np~===Note:=== if you are running a soil sample, you will need to use glass wool or a cottonball, either in the glass rod or top of exetainer vial, or in the glass cap of the sample vessel.%%% %%%
#”Exetainer cap:” __wipe the threads of your sample vial until they look 100% clean__. Also wipe the threads on the cap attached to the glass rod. Put a thin, smooth coat of grease on both sets of threads, then carefully screw the vial onto the cap. Make sure the O-ring is sitting flat.%%%”Sample vessel:” __carefully wipe the groove on the sample vessel until it is 100% clean.__ Do the same for the grove in the glass cap, and the O-ring. Make sure there are no dirt or dust particles, and no hairs. Re-grease O-ring, and set it in the groove of the sample vessel. Clamp the glass cap onto the sample vessel.%%% %%%
#__Insert the glass rod or glass cap into the Ultra-torr, and tighten finger-tight.__%%% %%%
#__Submerge the sample vial with LN{SUB()}2{SUB} from the short blue dewer__. If you are using the exetainer set up, BE EXTREMELY CAREFUL NOT TO SPLASH ANY LN{SUB()}2{SUB} ON THE EXETAINER CAP (if you do so, it will freeze the O-ring and you will not be able to zero your sample).%%%Let the sample come to equilibrium with the LN{SUB()}2{SUB} (it will stop boiling), and make sure that the entire sample is submerged.%%% %%%
# __When the sample has reached equilibrium, open the valve for the station, and let the station zero to its original zero-point-o-the-day. __ When it reaches that point, __close off the station valve, and check that there are no leaks in the vacuum.__ If the station is leak-free, then you now have a closed system between the sample vial and the collection tube, and it is under vacuum pressure.%%% %%%
#__Remove the LN{SUB()}2{SUB} from the sample vial, and set aside. Place the sample vial in a beaker of hot (boiling) water, and the collection vial in one of the silver dewers.__ The hot water should entirely cover the sample in the sample vial. If you are using the exetainer set up, be sure that the water is not touching the exetainer cap. The LN{SUB()}2{SUB} should just barely touch the bottom of the collection vial. This will allow the water from the sample vial to collect at the bottom of the collection vial, and will not condense in the Ultra-torr region.%%% %%%
#__Write down the start time of the extraction for this sample__ (= the time when the sample is HOT and the collection vial is COLD).%%% %%%
#__Label a piece of lab tape with the sample ID, and stick it on the manifold above the correct station.__ %%% %%%
#Let the sample run for at least as long as designated for its sample type (see beginning of protocol).%%% %%%
#Once the sample is on, __repeat this protocol for the remaining stations__. It’s best to go in order so that you don’t get confused.%%% %%%
#During the hour that the sample is extracting, make sure to __keep a watch on fluid levels__. You want to keep the LN{SUB()}2{SUB} touching the bottom of the collection vial, and the hot water covering the sample. Also make sure that the water trap is topped off with LN{SUB()}2{SUB} at all times.

===__Removing the Samples__===
#__Check the line of the station and for condensation.__ If you see any, then there is a problem. Follow the solution for problem ~np~#~/np~2 in “Troubleshooting” below.%%% %%%
#__Remove the collection vial and wrap Parafilm around the top__.%%% %%%
#Remove sample label from the line, and place it onto a plastic tube. Invert the plastic tube onto the collection vial. Let the ice melt.%%% %%%
#__Remove tube with soil or plant sample and return sample in original sample vial __(unless there is more of the sample left, in which case you can discard the extracted sample). __Wash the tube with a kimwipe and acetone (~~royalblue:best~~) or ethanol.__%%% %%%
#__Replace a clean, dry collection vial in the station.__%%% %%%
#You are now __ready to start another sample.__ Begin at step 3 of “Running your samples”.%%% %%%
#When the ice thaws in your collection vial, transfer the liquid to the respective plastic vial, and cap off.%%% %%%
#Wash the collection vial with acetone or ethanol, and place inverted collection vial in the rack to dry.%%% %%%
#__Pipette your samples into the HD or {SUP()}18{SUP}O vials.__ If you can, use 200μl for {SUP()}18{SUP}O and 30μl for HD.%%% %%%
#__Store your water samples in the -20 C freezer in a labeled bag or box with your name and contact information.__

{img fileId=”147″ thumb=”y” alt=”” rel=”box[g]”} {img fileId=”148″ thumb=”y” alt=”” rel=”box[g]”}

!!Other resources
!!Notes and troubleshooting tips
__”~~royalblue:Problem 1~~”: Can’t get sample to zero at initial zero-point-o-the-day (although station zeroed fine before)__
===Reason:=== Some water vapor, dirt or debris is preventing the pump from pulling or maintaining a vacuum.
”===Solution:===” This is a bit tricky to fix once everything is set up. What you need to do is remove the sample vial (or vessel) from the line, and cover the top with parafilm. Keep the entire sample submerged in LN{SUB()}2{SUB} (it may help to wedge the vial into the middle of the large forceps to keep it from falling in the LN{SUB()}2{SUB}). Carefully clean the glass cap and O-ring (if you are using the sample vessel) or the exetainer cap (for the exetainer set up), and re-grease the necessary articles. When you are sure everything is VERY clean, then you can remove the parafilm from your sample vial (or vessel) and clean the threads of the vial, or the top groove. Then quickly put the sample back on the line, freeze it, and start the vacuum again (start with ~np~#~/np~7 of “Running your samples”).

__”~~royalblue:Problem 2~~”: Water is condensing inside the line of 1(+) station(s).__
===Reason:=== Your sample is overloaded with water, and it’s clogging up the system.
===”Solution”:=== There is no great way to deal with this other than to use smaller samples – water condensation means you’ve overwhelmed the system with liquid. The best thing you can do is to leave everything attached, so that the vacuum is maintained, and use a hairdryer to coerce the condensation from the sample side to the collection side. Continue to move the condensation to the collection vial until you can no longer see any. Then you can remove your collection vial.

!!Links to resources and suppliers

!!Literature references

!!Health, safety & hazardous waste disposal considerations
PLEASE remember to wear safety glasses at all times (NO EXCEPTIONS) and gloves when working with liquid nitrogen and the boiling water.

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