INTRODUCTION

This document describes how to prepare water samples for analysis of oxygen and hydrogen isotopes by cavity ring-down spectroscopy (CRDS).  Also included are instructions to clean used sample vials for reuse.  The method is intended for analysis on the Oceanography Picarro but is applicable to the IsoLab instruments as well.

SAFETY

This is probably the safest protocol used in the lab.  There are no hazardous materials whatsoever.

MATERIALS
Note: Supplies can be found in the “Water Isotopes” drawer along the east wall.

1. 100-1000 μL pipette set to 250 μL
2. Dry pipette tips
3. Numbered 300 μL GC vials (dry) and new caps
4. Cryo-box for GC vials or blue tray
5. Empty tall beaker to collect used pipette tips

STANDARDS
Currently the HEEL uses the following IsoLab standards:
Name         Accepted δ18O (‰)        Accepted δD (‰)
BW                         -20.01                                 -156.87
SW                         -10.55                                 -75.63
KD                           0.00                                     0.65
Note: Shake standard bottles vigorously for 5-10 seconds before opening to isotopically re-equilibrate the water vapor and liquid water. 

MEMORY TEST
If the instrument has been idle or used for seawater previously check the memory of the system by running standards with large isotopic contrasts back-to-back.  This will tell you the proportion of the current sample derived from the previous sample.  Five to ten percent memory for δD is ideal but 10-15% is more typical for the Oceanography instrument.  If the memory is 15% or more consider cleaning the vaporizer.

1. Load eleven vials with the following standards in the following order: DI water (conditioner), BW, BW, SW, SW, KD, KD, SW, SW, BW, BW.
2. Start a run (see Analysis by CRDS protocol).  Set the number of injections per sample to 10. The run will take about 21 hours to complete.
3. Calculate the memory for each sample as:
   • range = absolute value of the mean from the last two injections from the current vial (j) minus the mean of the last two injections from the previous vial (i)
   • remaining = absolute value of mean of the last two injections of current vial (i) minus first injection of the current vial
   • memory (%) = remaining / range * 100

SETTING UP A RUN
Runs start with two DI water conditioners, a full set of standards, and the unknown samples.

Conditioners both warm up the instrument and are used to transition between two samples or standards that differ widely in their isotopic values (>10‰ δD and > 2‰ δ18O).  With large isotopic differences it can take 5 or more injections to clear the instrumental memory.  Conditioners make this transition but are removed from any calculations later on.

Typical runs have three reference standards: two that bracket the sample range and a third for validation.  Standards are run in duplicate in consecutive tray positions.  The first of the two should be identified as a “Conditioner” and will be excluded from the calculations.  Standard sets are repeated every 15-20 unknown samples.

Unknown samples should be ordered by their expected isotopic values (either increasing or decreasing) as best as possible.  If expected values are not known a test run of all samples is advised.  As with the standards, duplicate samples can be added anywhere in the run where large differences in isotopic values between two adjacent samples is expected (or you don’t know).  Identify the first as a “Conditioner” to exclude it from the analysis.

An example run sheet can be found here.  The sampleType column identifies specific vials as “Conditioners”, “Standards”, or “Samples”.  These are the only allowable options for the R code.  trayNum and vialNum are the tray position number and vial number respectively and must be integers.  sampleDesc identifies the sample or standard.

LOADING VIALS

1. Array samples in order they are to be run.
2. Grab a dry vial from the “Dried” beaker and record vial number, sample identifier, and sample type (Conditioner, Standard, or Sample) on the run sheet.
3. Place dry pipette tip on pipette
4. Vigorously shake sample or standard bottle for 5-10 second to re-equilibrate the water with the vapor.
5. Open sample bottle and pipette 250 μL into the vial.  Recap sample bottle.
6. Cap vial with a new cap.  It should be very snug but not so tight as to distort the septa.
7. Eject pipette tip into empty beaker.
8. Place loaded vial in either a cryo-box or rack.  Be sure to maintain the run order.
9. Repeat 2 through 8 for the full run.  The same pipette tip can be used for duplicates of the same standard or sample. When finished loaded vials should be stored in the refrigerator.

PREPARING USED VIALS AND PIPETTE TIPS FOR REUSE

1. Remove and throw away the cap.  Shake leftover water into the sink.
2. If salt or sediments are present rinse with DI water (you will likely have to pipette water into the vial).  If necessary, wash with Liqui-Nox soap and rinse minimum 3 times with DI water.
3. Place vials into a clean beaker and cover loosely with aluminum foil.  Do the same for used pipette tips.
4. Place in the 60 ˚C oven in FSH 333 for ~4 hours.  When done, leave covered and label that they have been dried.  Place in “Water Isotopes” drawer on east wall.