Stanford University

This instruction sheet is to aid in the measurement of trace levels of Hg in environmental samples. The use of CVAFS (Cold Vapor Atomic Fluorescent Spectrometry) is essential for Hg, it and Cold Vapor Atomic Absorbance Spectrometry are the only methods that are accepted by the EPA and the general Hg community for Hg analysis. Since the CVAFS uses a fluorescent signal instead of an absorption signal the detection limit is significantly lower and also allows for greater reproducibility. Refer to EPA method 1631 for a more detailed outline of the technique.

The theory of the method is that solid samples are digested, usually with Aqua Regia, to release Hg into solution. The solution is then preserved with a very strong oxidant (BrCl, bromine monochloride) in order to keep Hg in solution as Hg²⁺, since it will not volatilize like Hg^o will. Water samples are also oxidized with BrCl. The purpose of this is to oxidize all the Hg (Hg^o, HgCl₂ complexes Hg⁺, Hg bound to organics, Hg bound to minerals, etc.) in the sample to Hg²⁺. The machine then takes the sample with Hg²⁺ and reacts it with a very strong reductant, Stannous Chloride (SnCl₂), to produce Hg^o, which is then stripped from the solution as a gaseous phase with Ar gas. Since Hg amalgams with Au, the machine uses Au coated quartz sand to trap the Hg. The Au is then heated and pushed onto a second Au trap. The point of these traps is to concentrate the gaseous Hg so that when the Hg reaches the analyzer it is a spatially small concentrated pulse of Hg. The analyzer is a UV source that causes fluorescence of the Hg in the analyzer chamber, which is then detected using a photo multiplier tube.

The Tekran 2600 CVAFS is a high precision instrument that is capable of running numerous samples quickly and accurately. The general working range for the machine is 0.5-100 ng/L, but the machine is capable of measuring 0.1-400 ng/L (samples above 100 ng/L should be diluted to avoid carryover). The machine uses about 35mL of solution (use either 60mL centrifuge tubes, not recommended or 40mL borosilicate I-chem vials with Teflon lined lids, highly recommended) and is an automated process taking ~4 minutes per sample. Though the machine is automated it should be checked periodically, once every 45 minutes or so. When something goes wrong with the machine it goes wrong fast and very badly. If anything goes wrong, call me immediately on my cell (650) 804-6995.

Generalized instructions: There will be a spreadsheet on the machine that needs to be filled out every day to monitor the machine. Please fill it out every time you run.

Machine startup and general running

1. ALWAYS use nitrile gloves, NEVER, NEVER use latex, vinyl, or rubber gloves when working with Hg. Latex and rubber provide absolutely no protection against Hg, you might as well not be wearing gloves if you use latex or rubber.

2. Samples to be run must first be preserved with BrCl at least 1hr before analysis, preferably 1 day or more of oxidation time (Refer to EPA method 1631 for BrCl recipe). BrCl is usually added at a concentration of 0.5% (v/v), but this depends on the amount of DOC in the sample. If there is a lot of DOC the BrCl reacts with it and fails to oxidize all of the Hg in the sample. BrCl at 1% is usually enough for all samples, there will be a slight yellow color to the sample when enough BrCl is added. Try not to exceed 2% BrCl, BrCl tends to get contaminated with Hg after awhile but at low concentrations (2% or lower) the added Hg is negligible or at ~2% is easily subtracted off as background. Note: ALWAYS MAKE and use BrCl IN A FUME HOOD!!! When making BrCl solution add KBrO₃ very slowly, about a pin-sized amount at a time, more than this can lead to boil over (reaction is very exothermic) and it also releases large amounts of Br₂, Cl₂, and BrCl gas. Any sample containing BrCl must be reacted with Hydroxalamine hydrochloride (HAH) before running it through the Tekran, failure to do so can severely damage the Au traps and the machine, consult EPA method 1631 for the recipe, HAH will be provided in the lab so you should not need to make any.

3. Turn on Ar gas tank, Ar gas needs to be ultra high purity (5.0) and the tank can never have contained He, VERY IMPORTANT, if He was ever used in the take the residual He significantly decreases the life span of the instrument lamp. Power up the machine, all the machines are run through the same power strip, all that needs to be done is turn on the power strip and all of the machines should turn on at the same time. Note: Computer is also hooked up to the power strip so the computer can only be on when the machine is on, this is not a problem usually you keep the machine on over night to get the lamp sensitivity to stabilize.

4. The main reagent for the machine is 3% Stannous Chloride solution (SnCl₂). For this reagent there is a specific 2L Pyrex jar for the SnCl₂ solution, the reagent is the only thing that goes in this bottle. For 2L of reagent (good for about 10hrs of analysis) measure out 60g of SnCl₂ and add to the bottle. Using a graduated cylinder that is only for use with the Tekran, fill to 230mL with DI water and then add 20mL of trace metal grade HCl. Add this solution to the 2L bottle and swirl until the crystals are mostly dissolved. Fill bottle to the glass ring on the outside of the bottle (2L mark) with DI water and shake bottle to dissolve the rest of the SnCl₂. Purge the bottle with Ar gas for a minimum of 1hr (overnight preferable).

5. While SnCl₂ is purging make the standards for the day. Standards used are generally as follows: 0.5ng/L, 1ng/L, 2ng/L, 5ng/L, 10ng/L, 20ng/L, 50ng/L, and 100ng/L, 0.1ng/L can be attempted but usually is difficult to do, >100ng/L (up to 400ng/L) can be done but not recommended due to carryover. Recipe for standards are included on attached spreadsheet, tertiary standard (denoted 3^o) is stored in the refrigerator has a concentration of ~1ug/L (Careful with the standard bottle it is glass). Note: Do not use 2^o standard and do not use all of the 3^o, need to cross reference it every time a new 3^o standard is created. If 3^o is running low let me know and I will make some more. There is a specific distilled water bottle either on the machine or in the fume hood for the Tekran only, please use this bottle only for applications dealing directly with the Tekran. To try and eliminate any unknown contamination try to use this bottle so there is consistency.

6. Before running samples remove phase separator rod from housing (be Very, Very careful the rod and housing are delicate and very expensive) and clean the rod with either methanol and wipe with Kimwipe. Replace rod and tighten until snug.

7. Put tension on all the lines of the pump. DO NOT overtighten, the tabs should all be at only about 45^o.

8. Check under the counter that the water reservoir is at least 2/3 full, NEVER let the water reservoir get below 1/3 full, very bad things result if this happens.

9. Check to make sure that there is plenty of room in the waste container, the machine produces about 1L of waste per hour of running.

10. Once steps 6 and 7 are done flip the switch on the pump unit from off to local and make sure that the water flows evenly down the phase separator wand and that the wash station fills up.

11. Start up the computer and open the Tekran software. Go to worksheets menu and click run. Create either a new worksheet or use one that you have previously used.

12. Initialize the machine by clicking on the Init button on a secondary table.

13. Enter 223 for the model number and 112 for all 4 of the rack Ids.

14. Once machine is initialized add in sample name and sample tubes. Add into sample ID section this set of samples. 3 cleans, 2 washes, 1 BrCl clean, wash, 1 BrCl clean, 1 wash, 2 DI blanks (have BrCl and HAH equivalent to your samples), 1 wash, and then the standards. BrCl cleans are done with 10% BrCl (v/v) with 4% HAH added.

15. Before any sample with BrCl is run HAH must be added to neutralize any free halogens, which can severely damage Au traps. For a 40mL sample preserved with 0.5% BrCl (v/v), 0.2% (v/v) of HAH needs to be added (generally 10-15 minutes before sample is run). This ratio is proportional, if 1% BrCl is used 0.4% HAH needs to be added or if 20mL of 0.5% BrCl sample is added to 20mL DI water (40mL total) then 0.1% HAH is needed. Important: Too little HAH is a problem, but too much is not nearly as bad. If you require 0.005mL of HAH, then add 0.01mL HAH because that is the lowest most pipettes go to.

16. Remove front panel of autosampler case and load samples, sample positions start at the far back left and move forward. Remember that Clean and Wash commands do not require a sample position so samples will start with BrCl cleans. Replace front panel and switch the pump switch from local to remote.

17. On the computer click configure run, select which samples you want to run, click OK, and then start run

18. When running, if there is a problem with the run and you need to stop it click stop run, this will stop the run at the end of the sample that is being run. Try not to click abort run, it has a bad tendency to crash the program. Also, try not to click add sample if you are adding samples wait until the run is finished and click configure run and set for the new samples or click stop run and then adjust for new samples in the configuration window.

Machine shutdown

1. When the days run is over run 2 washes, 2 BrCl washes (same as above), and then 2 more washes. If things are really dirty in the lines or the phase separator a wash or two of 5% Aqua Regia may be used, but do not use more than 5%. Always end a run with at least 2 washes regardless of the cleaning solution used (BrCl or Aqua Regia).

2. After cleaning swap the SnCl₂ reagent with the 2L bottle of DI water. Flip the switch on the pump from remote to local and let run for 15 minutes. If this is not done then SnCl₂ will start precipitating as SnO in the lines and all of the lines will have to be replaced.

3. Once 15 minutes have passed flip the pump switch to off. THIS NEXT STEP IS A MUST!!! Make sure you release the tension on the sample line (clear tubing in the pump), the other tubes may also be released but the sample one must be done or irreparable damage can be done to the tubing and it needs to be replace (not cheap). Power off the machine and turn off the gas. After a few days of running 2-10 the sodalime trap needs to be replaced, please notify me if this needs to be done.
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