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CRDS Water Isotope Analysis

Introduction

Cavity Ring-Down Spectroscopy (CRDS) is an optical absorption technology capable of estimating the amount of individual isotopologues of several gas species. In our case, we are using CRDS for water vapor isotopologue measurement. We have several CRDS instruments manufactured by Picarro and this document aims to help you use them to analyze your water samples. You can read more about the technology from Gupta et al. (2009) and Steig et al. (2016).

At the moment we employ four CRDS instruments, two L2140-i, one L2130-i, and one L2120-i and the way they are used will depend on the peripherals attached to the instrument. We have two main types of autosamplers for discrete water sample analysis: PAL [blah] and [blah]. We also have two continuous flow analysis systems: ice core melting CFA and atmospheric water vapor analysis. This document focuses on the discrete analysis but you may need to reference various sections below when switching a CRDS box from CFA to discrete.

The CRDS instrument names are Mildred (L2140-i - dD, d17O, d18O), Phoenix (L2140-i - dD, d17O, d18O), Abel (L2130-i - dD, d18O) and Dr. DeSoto (L2120-i - dD, d18O). We have also historically had Gorky (an L1102-i) which is now on an extended sabbatical. The autosamplers we have are LeapTech LC-PAL, LeapTech GC-PAL, and a [blah blah]. The CRDS instruments seem to play musical autosamplers frequently enough that you will need to refer to appropriate sections below depending on what autosampler is attached to what CRDS box.

For clarity, sections specific to the Leap Technologies PAL autosamplers will have a PAL tag while the Picarro ALS-G autosampler will have a ALS-G tag.

Safety

Users must be careful with syringe needles and glass vials. If either of these items are no longer useful (i.e. broken), they must be discarded into the broken glass container. The vaporizer is 110 °C and the septum nut can be hot to touch.

Terse Procedure - TL;DR

  1. Clean up previous run – close the Coordinator software, remove existing vials.
  2. Load new waters - load 200 µL of water into 300 µL fused insert vials.
  3. Tray description - write the contents of each vial in a notebook or enter them directly into a tray description file.
  4. Prepare autosampler - put the vials in appropriate tray, clean the syringe, modify the job, don’t change the septum yet.
  5. Daily log - Complete each field in the daily log.
  6. Start Run – Start autosampler, open Coordinator Launcher, select injection method, click Change Septum, wait for first injection to complete.
  7. Change Septum – when the Coordinator indicates the first injection is complete, change the vaporizer septum and press “Septum Changed” when done.
  8. Tray Description – create a tray description csv file and save it as S:\Data\projects\[instrument]\TrayDescriptions\yyyymmdd_[sample-set-ID]_TrayDescription.csv.
  9. Data - use Python, R or Matlab to process your data.
  10. Cleanup - empty the existing vials, throw the caps in trash, discard water down the sink, put vials in a beaker in the drying oven, clean vials if needed.

Exhaustive Protocol youtube icon

Clean up previous run youtube icon

Close the Coordinator...be patient, it will close eventually. Remove vials from the previous run and set them aside for later cleanup.

Atmospheric Air Selector Valve
Instruments may have a valve behind the vaporizer used to toggle between dry air for discrete injections or atmospheric air from either the lab or outside the building. The instrument is happier if it is measuring some amount of water vapor rather than bone-dry air. Use this valve to select between atmospheric air when the instrument will not be used for a while, or dry air, when you wish to analyze samples. The current position in this photo is dry air.

If the instrument was not previously running AND the water backgrounds are currently in the 10,000 to 20,000 range, the valve on the back of the vaporizer (see photo) is allowing atmospheric air to flow into the instrument. Rotate the valve 180° to allow dry air to flow into the instrument. You should see the water backgrounds begin to drop. Ideally, the water background is below 100 ppm before you start.

Load new waters youtube icon

Load 200 µL of water into 300 µL fused insert vials. The default is to load 200 uL but perhaps your project has limited sample and you choose to load less. In this case, load identical volumes for samples and standards. Use the pipette, pipette tips, and trays in 303B drawers labeled ‘Laser’. Vials are located on the lab bench above these drawers. You may also wish to load 1 mL of water. In this case, use the 2 mL vials without the 300 µL inserts which are located in a drawer labeled ‘Laser – extras’.

Use one full tray of dry clean pipette tips and also have one empty pipette tray available. For each distinct water, use a clean dry pipette tip, discarding the wet tips into the tray that started out empty. In this way, you will have one pipette tip tray with dry clean tips and one tray to receive the wet tips. If the pipette itself becomes wet, either on the inside or outside, ensure it is dry before proceeding. Use wipes or paper towels and / or compressed air to dry it out.

Make sure the caps on the vials are just tight enough but not too tight. If the cap septum is overly concave, it is too tight. Put all standard waters and all sample waters back in the refrigerator when loading is complete. Put the wet pipette tip tray in the drying oven. As you are loading waters, write the tray position number, the water type or sample ID in a notebook or enter them directly into a tray description csv file.

Lastly, when the 300 µL inserts are used, a tiny bubble is left at the very bottom of the insert after pipetting your water. This bubble must be dislodged. Hold the cap tight in one hand and flick the bottom of the vial with your other hand to jostle the bubble loose. Assume this is to be done with every single vial.

Prepare autosampler

Choose autosampler - We frequently play musical autosamplers and so before you can proceed you must decide which autosampler is attached to your instrument. For clarity, sections specific to the Leap Technologies PAL autosamplers will have a PAL tag while the Picarro ALS-G autosampler will have a ALS-Gtag. Nearby photos may help you decide which autosampler you have.

LC-PAL Autosampler
LC-PAL Autosampler.
ALS-G Autosampler
ALS-G Autosampler.

Vials - Some of the autosamplers require use of a metal tray while others allow for the blue plastic trays. If you used blue plastic tray(s) as a temporary loading tray of samples and standards transfer your vials to the metal tray on the autosampler. If you moved the metal tray at all or are placing blue plastic tray(s) on the autosampler tray holder, make certain it is properly seated. Visually check the caps one last time; no cap should be overly concave or crooked.

ALS-G syringe attachment
ALS-G Use the black toggles to remove / install the syringe into place. The arrow indicates a place where the tip of the needle can become lodged. Make sure the needle tip extends through the hole.
PAL syringe attachment
PAL Use the red thumb nut and the toggle to remove / install the syringe into place. Similar to the ALS-G autosampler, mind the area near the tip of the syringe to ensure it does not become lodged.

Remove the syringe - Whether you are going to clean the syringe or replace with a new one, you will need to remove the existing syringe. You can either just remove the syringe where the autosampler resides (preferred) or press "Chg Syringe", on the autosampler control software. CAUTION - Often times pressing "Chg Syringe" seizes the plunger in the barrel of the syringe. If you did press "Chg Syringe", you will need to press "Swap done" when you are finished. Remove the syringe from the autosampler by adjusting both black toggles, one at the plunger and one at syringe-body. Use the photo to help you identify the toggles. The top plunger toggle twists while the bottom syringe-body toggle pulls out and twists.

Clean the syringe - Find the syringe cleaning kit. Fill the 'helper' syringe that is in the kit (not the one you just removed in the above step) with the DI water from the little vial and set aside. Place the syringe you just removed from the autosampler into the DI water vial and pull the plunger up half way or as far as you can without forcing it. Use the 'helper' syringe and apply DI water to the top of the autosampler syringe (e.g. where the plunger meets the syringe glass body - see photo). Set the helper syringe aside. Continue moving the autosampler syringe up and down while it is in the DI water vial until plunger can be removed from syringe body (this usually takes a while and you must be patient and gentle). Wipe off plunger with a paper wipe. Reinsert the plunger into the body. If you can’t remove the plunger during cleaning, break it, or it was already broken, obtain a new 10 µL syringe from the drawer labeled "Autosampler Syringes" to the right of the fume hood in 302A and near the IsoLab office. Discard the old syringe to the broken glass box in 303B under the northernmost sink (since this is not a skin piercing style needle, the broken glass box is sufficient). You do not need to clean a new syringe.

Picarro syringe cleaning
The Picarro syringe cleaning kit has everything needed to clean a syringe, except wipes. It also has the septum for the vaporizer as well as the large needle which may be helpful in changing the septum.
Picarro syringe cleaning
Using the Helper Syringe to apply DI water to top of to-be-cleaned syringe.
ALS-G autosampler software
Use the ALS-Gautosampler software to declare the number of vials and injections per vial for your current run.
Dr DeSoto autosampler controller
Use the PAL autosampler control pad to edit the job (e.g. number of vials, number of injections per vial)

Autosampler jobs - Modify the autosampler job according to your current number of vials and your preferred number of injections per vial. Here again, you will need to choose which instructions to follow depending on the autosampler you are working with.

Daily Log youtube icon

The lab uses a daily log for each instrument or preparation line to allow users a first glance at the readiness of the instrument. By comparing the current state of the instrument to historical states, you are more informed about the instrument and whether or not it is functioning properly and ready to run your samples.

Each daily log is web based and browser accessible. No link is provided here by design. Open the browser on the controlling computer and you should see at least two tabs already open. One tab is this SOP and the other is the daily log. If the browser has more than two tabs open, it may have additional SOPs. Use the bookmark toolbar as needed if tabs have been closed.

Work through each cell of the daily log. If you are uncertain where to find certain information, hover over the column header tip, denoted by a ⓘ symbol.

Injection Method - As part of the Daily Log, you will enter the injection method. This is chosen from a drop-down menu you will see immediately after launching the Coordinator software. Selections like High Throughput, High Precision, or O17-High Precision may be available. These modes of analyzing samples are aptly named. High Throughput mode aims to analyze many samples quickly and each injection takes ~5 minutes. High Precision mode aims to obtain better precision and injections take ~9 minutes each. O-17 High Precision aims to achieve the highest precision and takes ~14 minutes per injection. The difference among these methods is the duration of time the instrument spends measuring and cleaning out each injection.

Vaporizer Septum - You will also be asked to indicate if you have replaced the vaporizer septum with a new one. Unless you have good reason not to, you will replace the old septum with a new one after you start the run below.

Make certain to press the 'save to log' button when you are finished entering data.

You are welcome to make notes if you have observed something with or done something to the instrument and would like to document that information. Use the "insert note" link at the top of the daily log to make a note. You may enter notes at any time.

Abel injection type selection
After you have opened the Coordinator Launcher software, you are faced with this injection type selection.
Abel Change Septum Button
Click this "Change Septum" button then wait until prompted to change the vaporizer septum.

Start Run youtube icon

Click "Run" on the Autosampler Control software and wait for the autosampler to proceed through a series of initiation steps. Next, open the Coordinator Launcher from the shortcut on the desktop and select High Precision or High Throughput. Click "Change Septum" in the upper right corner of the Coordinator window and wait for first injection to complete.

Open the Coordinator Launcher, select High Precision or High Throughput, then select Start on the Autosampler (F4 from Home screen). Assuming you are running only one tray, rotate the wheel until 'Selected' is highlighted and center click. If you have two trays, select 'All' with a center click. Click "Change Septum" (upper right corner of the Coordinator window) and wait for first injection to complete (~ 8 min 30 sec).

Tray Description youtube icon

A good time to create the tray description file is while you are waiting for your instrument to be ready to have its septum changed. The tray description file was historically used by the Coordinator software to assign each injection an Identifier which you give to each vial. However, since we no longer use the Coordinator data, the Python and Matlab scripts referred to below instead make use of this tray description file. As a template, use S:\Data\projects\[ instrument ]\TrayDescriptions\_TrayDescription.csv. Use a text editor to open one of these templates. If you double click on one of these templates, it will open in Notepad++. The header row in these templates is: "Project", "Tray","Vial","Identifier 1","Injections". The Project column should be your sample set ID and may be several sample set IDs if you are running multiple projects at once. The Tray and Vial columns are already filled in. Type in the water that is in each vial under Identifier 1. Example Identifier 1 entries are "SW", "WW", or "VW", without quotes. Type the number of injections under Injections. This may seem redundant, and hopefully it is, but sometimes when the autosampler fails or an injection is otherwise compromised, adjusting the number of injections in this file after the run has completed will help in data processing. Save this file as yyyymmdd_[SampleSetID]_TrayDescription.csv (where yyyymmdd is today's date, and [SampleSetID] is your project sample set ID and the square brackets are not included). The "TrayDescription" part of the filename is essential for some parts of the Python and Matlab code.

Change Vaporizer Septum youtube icon

Dr DeSoto vaporizer and septum
Picarro vaporizer and septum. The injection port where the septum is the be changed in shown with a red circle.
DrDeSoto septum change
The Swagelok nut, large gauge needle, and vaporizer septum.

After the first injection is complete, the Coordinator will indicate when it's appropriate to change the septum. Remove the magnetic cover and use the slotted wrench side of this cover to loosen the nut (see photo). Remove the nut with your fingers, taking care as it is hot. The septum should be stuck inside the nut. Use the large-gauge needle that is in the syringe cleaning kit to extract the septum (see photo). Discard the old septum into the trash. Place the new septum into Swagelok nut using the blunt end of large-gauge needle. Place the nut back onto the vaporizer, taking care not to allow the septum to fall out when you turn the nut over. Hand tighten the nut; you should feel the nut bottom out. Press “Septum Changed” which is the same button you pushed to begin changing septum in Coordinator window.

Data

The Picarro instruments provide data in at least four different levels described in Schauer et al (2016): "(1) coordinator data, (2) user data, (3) private data, and (4) spectral data. The coordinator data are what most users post-process to the VSMOW-SLAP scale and publish; the coordinator file contains a single row of data for every injection and is readily imported into a Laboratory Information Management System (LIMS) such as LIMS for Lasers 2015. Each datum in the coordinator data is the average, variance estimate, or otherwise-summarized value across the 120–500 s duration of usable data from an injection, and originates from the user data. The user data file is typically used if the pulse analysis fails and a user employs the pulse analysis software to redefine the peaks. The private data are high-resolution unprocessed data with all instrument parameters recorded at a frequency of approximately 1 Hz but are still a reduction of the spectral data. The spectral data are the measured absorbances during each ring-down and have a frequency of approximately 500 Hz".

There are several ways you can proceed with your data analysis: DIY, easy, or comprehensive.

Picarro vial cleaning contraption
Picarro vial cleaning contraption. The red arrows signify the importance of not turning the DI water on all the way as well as making sure the tubing is firmly pressed on the the DI faucet barbed fitting.

Cleanup

Vials

Take vials with previously analyzed water to the wet lab. Open each vial, discard the caps into the trash, and discard the water down the sink by gently shaking the vials. If the vials contained dirty or salty samples, they must be soaked in DI water. You will have to pipette DI water into each vial. You may also use the vial-cleaning-hose-pipette-contraption as shown in image. Once vials are clean, gently place vials in beaker and put them into the drying oven. If soaking in DI water does not get the vial inserts clean, use the glass pipette rubber hose tool attached to a DI water spout to force DI water into the bottom of the insert.

Vaporizer Cleaning

This procedure is completed irregularly depending on the memory or carry-over from sample to sample. More salt buildup inside the vaporizer will leak to more carry-over from previous samples. Picarro wrote up a procedure for this. A pdf of this procedure is located under Resources in Manuals. We have slightly different parts to assemble the apparatus but the general workflow is the same. Also, consider using 5-10% acetic acid as the first rinse of the vaporizer. Follow this by numerous rinses with 18 MΩ water.

Running more samples?

If you are not going to run more samples and you do not know of anyone after you wanting to run samples, rotate the valve on the back of the vaporizer (see photo above) 180° to allow room air to flow through the instrument rather than dry air. You should see the water backgrounds begin to increase.

Vials

Take vials with previously analyzed water to the wet lab. Open each vial, discard the caps into the trash, and discard the water down the sink by gently shaking the vials. If the vials contained dirty or salty samples, they must be soaked in DI water. You will have to pipette DI water into each vial. You may also use the vial-cleaning-hose-pipette-contraption as shown in image. Once vials are clean, gently place vials in beaker and put them into the drying oven. If soaking in DI water does not get the vial inserts clean, use the glass pipette rubber hose tool attached to a DI water spout to force DI water into the bottom of the insert.

Vaporizer Cleaning

This procedure is completed irregularly depending on the memory or carry-over from sample to sample. More salt buildup inside the vaporizer will leak to more carry-over from previous samples. Picarro wrote up a procedure for this. A pdf of this procedure is located under Resources in Manuals. We have slightly different parts to assemble the apparatus but the general workflow is the same. Also, consider using 5-10% acetic acid as the first rinse of the vaporizer. Follow this by numerous rinses with 18 MΩ water.

Troubleshooting

Suggested Reading

Other Resources

Picarro has a series of tutorial videos that may be helpful.

Signature

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

Last updated: 2023-01-23 16:01:39