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

Risks associated with this method: syringe needles, glass vials, hot surfaces.

Sharp edges - If you encounter a broken syringe glass body or metal needle, edges will be sharp. Don protective leather gloves while removing broken pieces. All broken items of this sort need to be discarded into the lab broken glass box located in 303B.

Hot surfaces - The vaporizer is 110 °C and the septum nut can be hot to touch. Wear protective gloves while handling the vaporizer septum nut.

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

Clean up previous run

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

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

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.

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.

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.

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.

• PAL - Press the F4 button on the autosampler control pad (see photo) to navigate to the home screen. From the home screen on the autosampler controller, turn the wheel to highlight the appropriate job and center click. 'First' should be 1, 'Last' should be the last vial in your tray (e.g. 54), 'Increment' should be 1, 'Count' should be the number of injections per vial you wish to have (e.g. 10). To change any of these values, rotate the wheel until the value is highlighted, center click, and turn the wheel to edit the number. Center click again when you are done editing. Press 'Home' (the F4 button) when you are finished editing the job.
• ALS-G autosampler job - Modify the autosampler job according to your current number of vials and your preferred number of injections per vial. If the autosampler control software is not open already, use the shortcut on the desktop to open it. Select "IsoLab" in the method field unless you have reason to pick a different type. The method is the way each individual injection is treated. Tray should always be "1". Start will typically be "1". End will be the position of the last vial. The #Inj is the number of injections per vial.

Daily Log

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

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

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.

Start Run

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

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

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.

• DIY - Of course you are welcome to take the raw data at any of the above mentioned levels and run with it.
• Easy and historical - This approach is easiest because it takes a single small file (produced by the Coordinator software) with only the most salient data and, using a Matlab script, provides you with water isotope values normalized to the VSMOW-SLAP scale. Coordinator data files are written to C:\IsotopeData\ in comma delimited value (.csv) format. Copy this file to your preferred project folder on the data server or to S:\Data\projects\[ instrument ]\raw\. Move to the North, South, or West Going ZaxThe naming of these three computers was inspired by Dr. Seuss' story about the North Going Zax and the South Going Zax (shown in the image). There is no West Going Zax in his story, but the same idea applies. computer. Open Matlab, type abel (or gorky, desoto, phoenix) and follow the prompts. After the script completes, the exported file is in S:\Data\projects\[ instrument ]\reduced\ or the reduced folder of your preferred project directory. NOTE, this approach is not compatible with the above below tray description file. You will need to create a tray description file without the project column.
• Comprehensive and recommended:

  These scripts are available on github.

  • picarro_h5.py - The picarro_h5.py script defines the run and combines all h5 file data into a single hdf5 file.
  • picarro_inj.py - The picarro_inj.py script finds all the injections and creates injection level data by summarizing (mean, standard deviation, etc.) the high resolution data within each injection or pulse.
  • picarro_vial.py - The picarro_vial.py script creates vial level data by summarizing all injection level data for all injections in each vial. While you may wish to go back and do more diagnostic analyses with the high resolution data or the injection level data, it is the vial level data that most will be interested in. The vial level data are calibrated to the VSMOW-SLAP scale using the included reference waters. A browser based report is created as a finale.

Cleanup

Troubleshooting

• The syringe has died - You come in to find the syringe mangled while the autosampler and instrument continue to run. Scroll up in the upper Coordinator window to find where the injections stopped working and note that vial number. Close the Coordinator. Replace the syringe with a new one. Discard dead syringe into the broken glass box located in room 303B under the northernmost sink (since this is not a skin piercing style needle, the broken glass box is sufficient). Modify the autosampler job to start on the vial you noted above. Start the instrument as normal, but don't worry about changing the septum.
• The water background is way too high - Check the valve on the back of the vaporizer to ensure it is pointing towards dry air (see photo at top of this page).
• Autosampler issue such as error or not moving. Both types of autosamplers have routine maintenance that may help. Ribbon cables within the autosamplers can also wear out with time. ALS-G Autosampler Maintenance - [need to write more here, for now, link to picarro docs]. Lubricant doc, Z-axis spring doc, Lubricant maintenance doc

Suggested Reading

Other Resources

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

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