University of Alberta

Digestion of Samples for Subsequent Total Mercury Analysis

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Determination of Total Mercury in Fish, Sediments and Vegetation: Sample Digestion Protocol

Written by Jane Kirk and Vincent St.Louis.

1.0 Scope and Application
2.0 Summary of Method
3.0 Safety Issues
4.0 Containers and Holding Times
5.0 Reagents and Standards
Quality Control
7.0 Procedure

1.0 Scope and Application:

1.1 Applicable Matrices: This method may be used to determine mercury in biological tissues, lake sediments, soils, etc.

1.2 Minimum Reporting Limit: The minimum reporting limit for this method is 5 ng/g (nanograms per g dry weight).

1.3 Dynamic Range: This method is designed for the measurement of total mercury (Hg) in the range of 5 - 5000 ng/g. The upper range may be extended to higher levels with the selection of a smaller sample volume.

2.0 Summary of Method:

A known weight of sample is digested using 7: 3 HNO3:H2SO4 (vol:vol) and heat. Bromine Monochloride (BrCl) is added to the sample container to oxidize all forms of Hg to HgII oxidation state. After a minimum of 12 hours the BrCl is neutralized by addition of Hydroxylamine Hydrochloride (NH2OH*HCl). Following neutralization, Stannous Chloride (SnCl2) is added to the sample to reduce the Hg from the HgII to the Hg0 oxidation state. The Hg0 is purged onto gold-coated glass bead traps (sample). The mercury vapour is thermally desorbed to a second gold trap (analytical) and from that detected by cold vapour atomic fluorescence spectrometry (CVAFS).

3.0 Safety Issues:

Specific safety concerns for each chemical can be found in the Material Safety Data Sheets for that chemical, all of which are located in the laboratory. Two extremely important areas of safety for this method are addressed below.

3.1 Chronic mercury exposure may cause kidney damage, muscle tremors, spasms, personality changes, depression, irritability and nervousness. Due to the toxicological and physical properties of Hg, only highly trained personnel using extremely cautionary procedures should handle high concentration standards. These cautionary measures include use of gloves and high volume hoods when preparing standards.

3.2 Strong, and sometimes hot, acid solutions are employed in the cleaning of equipment, preparation of reagents and in sample digestions. Proper acid handling techniques should be employed whenever acids are being used. These techniques include the use of acid resistant clothing, self-contained breathing apparatus/respirators, and the utilization of high volume fume hoods.

4.0 Containers and Holding Times:

4.1 Digestion vessels consist of Savillex # 561R2 teflon bombs. New bombs are cleaned by rinsing in tap water and immersing in concentrated reagent grade nitric acid heated to 65oC for 6-8 hours. Bombs are then rinsed in Milli-Q water and immersed in 30% Baker Instraanalyzed HCl heated to 65oC for 6-8 hours. Immediately following removal from the bath, the bombs are immersed in Milli-Q water and rinsed at least 3 times with Milli-Q water. They are dried either by air or in a drying oven, then bagged. Following digestions, Teflon bombs are scrubbed with a test tube brush using 5 mLs of Sparkleen in 1 L hot water. Bombs are then rinsed with distilled water and immersed in 30% Baker Instraanalyzed HCl heated to 65C for 6-8 hours. After acid washing, they are rinsed in Milli-Q water at least 3 times, dried by air or in a drying oven, and then bagged.

4.2 The bombs are tightened before the heating stages of the digestion using a Savillex #55 wrench set.

4.3 Once digested, samples can be held for refrigerated 2-5 days in the Teflon bombs before they are analyzed.

5.0 Reagents and Standards

5.1 Reagents: All reagents and/or dry chemicals used to make reagents must be of the highest purity available from the vendor and shown to be low in mercury. Upon receipt at the laboratory, containers will be marked with the date of receipt and stored in appropriate areas. When reagents are mixed for use in this method, the person who mixes them will initial and date the reagent container.

5.1.1 Milli-Q Water: Ultra pure reagent grade water shown to be >18 MW starting from pre-purified source (distilled, RO, etc.). The water is delivered through a 0.2 uM filter. All water is obtained from a Millipore ELIX 10/Milli-Q water purification system.

5.1.2 7:3 Nitric: Sulfuric Acid (HNO3: H2SO4) (vol:vol). Baker Instra-analysed HNO3 and H2SO4 acids are used. Each new batch of acid is analyzed for Hg content prior to its use in digestions.

5.1.3 Bromine monochloride (BrCl): Dissolve 27 g of reagent grade Potassium Bromide (KBr) in 2.5 L concentrated ultra trace HCl. Place a Teflon coated stir bar into the bottle and stir for 1 hour in a fume hood. Slowly add 38 g reagent grade Potassium Bromate (KBrO3) to the solution while stirring. CAUTION: This needs to be done slowly and in a fume hood because large quantities of free halogens are produced. As you add the KBrO3 to the solution, the color should change from yellow to red to orange. Cap bottle loosely and allow to stir for an additional hour. The BrCl is analyzed for Hg prior to adding to samples.

*To reduce the Hg content of the reagents, muffle both the KBr and the KBrO3 overnight at 250oC. Take the reagents out of the muffle furnace while still hot, and dissolve KBr in HCl as soon as possible.

5.2 Standards: Upon receipt at the laboratory or on the day of preparation, containers should be labeled with the date received or made and the initials of the person preparing them. The stock and substock standards should by stored outside the clean laboratory to prevent contamination of the entire lab.

5.2.1 Stock Standard (1000 mg/L): Commercially available Hg standard verified against a NIST standard reference material. All subsequent standards are prepared using the stock standard. Before preparing other standards, ensure the expiration date of the stock standard has not been exceeded.

5.2.2 Sub stock or Secondary Standard (1000 ng/mL): The stock standard (1000 mg/L) is diluted by a factor of 1000 with 1% Baker Instra-analysed HCl.

5.2.3 Spike standard (5 ng/mL): The secondary standard (1000 ng/mL) is diluted by a factor of 200 in a clean Teflon bottle. You may wish to make up a more concentrated spike standard (e.g., 10 ng/mL) if the samples that you are analyzing are very concentrated. The spike standard must be compared to the previous spike standard and agree within 5%. Prepare fresh every 6 months or when it goes off.

6.0 Quality Control

Each analyst hired in our laboratory must demonstrate the ability to generate acceptable accuracy and precision with this method. This includes the ability to recover and reproduce standard reference material concentrations, maintain acceptably low acid blanks, produce acceptable relative percent differences between replicates, and produce spike recoveries that meet acceptance criteria.

6.1 Digestions are carried out in batches of 60. A certain number of these bombs must be dedicated to quality control.

Acid blanks: In each batch of 60 digestions, 2 bombs are used for acid blanks. These are prepared by adding 7 mL 7:3 HNO3: H2SO4 (v:v) and tightening the bomb lids using a Savillex #55 wrench set so that they are wrench tight. They are then heated to 25oC for 2 hours in a drying oven. After cooling in a refrigerator, 19 mL of Milli-Q water and 1 mL of BrCl are added to the acid blanks. Bombs lids are retightened and put back in the drying oven overnight at 60oC. Acid blanks are analyzed to demonstrate freedom of reagent and digestion vessel contamination.

6.2.1 Acceptance criteria: The concentration of Hg in the acid blanks should always be three times lower than that of the samples being analyzed.

6.2.2 Corrective Actions: If contamination has occurred during the digestion process (e.g., contaminated 7:3 HNO3: 2SO4 (v: v) or digestion vessels), then the entire batch of samples must be rerun.

6.3 Standard Reference Materials (SRM): In each batch of 60 digestions, 2 bombs are dedicated to standard reference materials. An appropriate SRM is chosen based on the matrix of the samples that you are running. Between 250-300 mg of SRM is weighed out into the bomb using a clean stainless steel scupula and the digestion is carried out with the same reagents and procedure as described in 6.2.

6.3.1 TORT-2 lobster hepatopancreas - This is SRM is generally used when invertebrate samples are digested. Hg concentrations for TORT 2 SRM can range from 210 ng/g to 330 ng/g, although we like to maintain results that are within this range as well as being within 10% of each other on any given digestion day.

6.3.2 MESS-3 Marine Sediment - This is the SRM used when sediment samples are digested. Hg concentrations for MESS-3 can range from 82.0 ng/g to 100.0 ng/g, although as for TORT - 2, results must be within this range and be within 10% of each other from digestion to digestion.

6.3.3 DORM - 2 Dogfish Muscle and Liver - This SRM is generally used when fish samples are being digested. Hg concentrations of DORM - 2 can range from 4380 ng/g to 4900 ng/g, but we maintain values that are within 10% of each other and within this range on any given digestion day.

6.4 Duplicates: Two samples in every batch of 60 bombs are weighed out in duplicate and digested as described above in sec. 6.2.

6.5 Spike Recoveries: Two samples and two acid blanks in every digestion are spiked with a known amount of Hg spike standard. After analysis, spike recoveries are calculated and must be within the range of 90-110%. Analysis of spike recoveries demonstrates that sample digestion was complete, and matrix interference has not occurred.

6.5.1 Acid blank Spikes: Two bombs are left without sample in them and after the 7 mL of 7:3 HNO3:H2SO4 is added, a known amount of Hg spike standard (generally 5 ng/mL) is added to the bomb. The rest of the digestion process is the same as described above (sec 6.2).

6.5.2 Matrix Spikes: The two samples that are digested in duplicate are also weighed out an additional two times each. After the addition of the 7 mL acid mixture, a known amount of Hg spike standard (generally 5 ng/mL) is added to these bombs. The amount of Hg spike that you add is dependent upon the amount of Hg that you expect to be in the samples. The amount of spike added should be approximately one tenth of the amount of Hg in the sample alone. Acceptance criteria: Percent recovery for a matrix spike must fall between 90 and 110%.

% Recovery = ((Concentration of Spiked Sample Concentration That The Spike Added)/Concentration of Unspiked Sample) X 100 Corrective actions: If the percent recovery falls beyond the range of 90 to 110%, a second spike on that sample must be run. If percent recovery for the second spike falls beyond the range of 90 to 110%, the digestion conditions (e.g., amount of acid used) should be revised.

7.0 Procedure

7.1 Comments: The samples are collected using ultra clean sampling techniques.

7.1.1 Interferences Static may be a problem when you are weighing out freeze dried, homogenized samples such as invertebrates and leaf litter. To help combat this problem, use a static brush on the outside of the sample bag or vial.

7.1.2 Helpful hints: Protecting samples from contamination cannot be over emphasized when working with Hg detection limits in the parts per trillion range. Extreme caution must be used throughout the preparation, collection and analysis procedures to avoid contamination. Once the samples have been weighed out into the Teflon digestion bombs, it is important that they remain upright. If the bombs are tipped over, sample can get onto the roof or into the threads of the bombs leading to sample loss or incomplete digestion. During the two hour digestion period at 125oC, small amounts of acid may be lost even though the bombs are wrench tightened. Bombs with sample and the 7 mL of acid mixture added are therefore weighed before and after the 125oC two hour digestion period. This provides an accurate volume of acid lost during digestion.

7.3 Lab ware:

7.3.1 All-plastic pneumatic fixed-volume and variable pipettors in the range of 5 L to 1 mL.

7.3.2 Analytical balance capable of measuring to the nearest 0.1 mg.

7.3.3 Samples are weighed out using acid washed stainless steel scupulas or razor blades.

7.3.4 The 7:3 NO3:H2SO4 (vol:vol), Milli-Q water, and BrCl2 used in tissue digestion are all distributed using repipet dispensers.

7.3.5 Drying oven: The drying oven is vented into the main duct of a fumehood to remove any fumes resulting from the two hour 125oC digestion period. The drying oven must be kept clean and should be wiped down after each digestion to remove any acid residues left behind from the digestion.

7.4 Sample preparation (to be carried out by the owner of the sample):

7.4.1 Invertebrates: Freeze dry samples in whirl packs. Sort or pick samples to remove extraneous material such as insects, plant matter, rocks, etc. Grind samples with acid washed glass mortar and pestle until they are homogenous. Samples should be stored in acid washed scintillation vials or whirl packs and the mortar and pestle washed well with Milli-Q water and KIM wipes between samples.

7.4.2 Sediments: Sediments may be digested wet or dry. If they are to be digested dry, they are processed as above for the invertebrates. If they are to be digested wet, they are stored in whirl packs in a freezer.

7.4.3 Fish: Fish samples may also be digested wet or dry. If they are to be digested dry, they are processed as in section 7.4.1. If they are to be digested wet, they are stored in whirl packs in the freezer.

7.4.4 Litter Samples: Freeze dry samples in whirl packs. Grind samples using an acid washed stainless steal coffee grinder or a stainless steal rasp. Samples should be stored in acid washed scintillation vials, whirl packs or ziplock bags and the grinder or rasp should be rinsed well with Milli-Q water and dried with KIM wipes between samples.

7.5 Sample Digestion:

7.5.1 Samples are weighed out directly into acid washed and completely dry Savillex #561R2 Teflon bombs. Bombs are dedicated as acid blanks, acid blank spikes, and matrix spikes at this time. SRMs and sample duplicates are also weighed out. A stainless steel scupula is used to transfer freeze dried and ground samples into the bombs whereas a razor blade may be used to cut off chunks of a fish tissues. The scupula or razor blade is rinsed with Milli-Q water and dried with KIM wipes between samples. Amount of sample used: The amount of sample that is weighed out depends upon the type of tissue being digested and the expected Hg concentration in the sample. If too little sample is used, you will not get a detectable level of Hg upon analysis. However, if too much sample is used, digestion may not be complete. For freeze dried invertebrate or fish samples, between 30 60 mg of sample is digested. For wet weight fish, generally between 150 - 250 mg of tissue is digested, however, smaller amounts can be used if there is a limited amount of sample available. For sediments, between 150 - 250 mg of wet sample, and between 60 - 125 mg of dry sample, is digested. When using wet sediments, it is important to insure that samples are well mixed and homogeneous prior to subsampling. For litter samples, generally between 60 - 125 mg of dry sample is digested.

7.5.2 Add 7 mL of 7:3 HNO3: H2SO4 (vol:vol) Baker Instra-Analyzed acid. This addition should be carried out in the fume hood and the bomb lids should be opened carefully so that no sample is lost (of special concern if dry samples are static).

7.5.3 Add the Hg spike (5 ng/mL) to the two spike samples and the two acid blank spikes. Spikes should be added to the bombs one at a time, pipette tips flushed prior to spike addition, and pipette tips changed between samples.

7.5.4 Tighten bomb lids using Savillex #55 wrench set so that they are wrench tight.

7.5.5 Weigh each bomb to get a pre-digestion weight.

7.5.6Put digestion bombs in clean drying oven at 125C for 2 hours.

7.5.7 Turn off the oven after the two hours and let bombs sit for a few minutes. Open oven door and let the bombs sit and cool down for ten minutes before handling them. Wear a lab coat, double gloves, and full-face shield to open the oven door as the acid in the bombs is under high pressure.

7.5.8 Remove digests from oven carefully. Wear lab coat, double gloves, and full-face shield to remove the bombs.

7.5.9 Transfer bombs to the refrigerator to cool and depressurize.

7.5.10 Reweigh bombs to determine how much acid was lost during digestion at 1250

7.5.11 Open bombs slowly and upright in fume hood by loosening the lids with Savillex wrenches. It is important that the bombs be completely cool at this point.

7.5.12 Gradually add 19 mL Milli-Q water to each digestion bomb in fume hood using a repipet dispenser. As this is a water to acid addition, rapid addition of the water is not advised.

7.5.13 Add 1mL of BrCl2 to each bomb using a repipet dispenser.

7.5.14Wrench tighten each bomb.

7.6 Sample Analysis: Samples are analyzed by cold vapour atomic fluorescence following total mercury analysis protocol (See Determination of Total Mercury in Water by Oxidation, Purge and Trap, and Cold Vapour Atomic Fluorescence Spectrometry" Protocol). 1 mL of tin chloride reagent is added to the bubbler followed by hydroxylamine hydrochloride, and then a subsample of the digested sample from the bomb. Between 50 uL and 3 mL of digest is added to the bubbler, depending upon the Hg concentration in the sample. 24 uL of hydroxylamine hydrochloride is typically used. The bubblers will be cloudy after sample addition as the acid reacts with excess tin chloride to make a tin colloid. Soda lime traps should be changed every day rather than every other day due to the high amount of acid in the samples.
Last Modified: 2002-11-05