Modified by David Kelly and Vincent St.Louis Original version written by Mark L. Olson and John F. De Wild, Wisconsin District Mercury Laboratory, U.S. Geological Survey, Madison, Wisconsin. This protocol has been listed here with permission of the original authors.
1.0 Scope and Application
2.0 Summary of Method
3.0 Safety Issues
4.0 Reagents
5.0 Quality Control
6.0 Procedure
7.0 References
1.1 Applicable Matrices:
This method may be used to distill filtered or unfiltered water samples. Application to tissues and sediments is discussed in an accompanying protocol.
1.2 Minimum Reporting Limit: Not applicable.
1.3 Dynamic Range: Not applicable.
Direct ethylation of natural waters has been proven to result in the release of only "reactive" MeHg, which represents only 5-60% of the total MeHg (Bloom 1989). The distillation procedure outlined is this document is required to remove matrices that may interfere with the ethylation process. The samples are distilled at 135oC with the addition of potassium chloride (KCl), sulfuric acid (H2SO4), and copper sulfate (CuSO4).
See also the accompanying protocol. Before beginning any of the procedures involved in this method, each individual must read and sign the Chemical Hygiene Plan developed for the lab. 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.
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 appropriate gloves and high volume hoods when preparing standards.
3.2 Strong acid solutions are employed in the cleaning of equipment and preparation of reagents. Proper acid handling techniques should be employed whenever acids are being used. These techniques include the use of acid resistant clothing and the utilization of high volume fume hoods and are discussed in detail in the Analytical Protocol.
3.3 This procedure requires the heating of a metal block. Direct contact with the block will result in serious burns. The transfer of liquid from the distillation vessel to the receiving vessel is in the form of steam. Occasionally leakage from the distillation vessel or pressure build up from transfer line blockage occurs. The analyst is required to remove the hot vessel from the block and readjust the cap in both these instances and must practice extreme caution while performing this task. Failure to remove the still first will result in hot and potentially corrosive liquid from the still rapidly back-flowing into the flowmeter or jetting out a disconnected line contaminating the sample and flowmeter and endangering the analyst.
4.1 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 the appropriate areas. When reagents are mixed for use in this method, the person who mixes them will initial and date the reagent container.
4.1.1 Reagent 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 A10 Gradient
water purification system.
4.1.2 Sulfuric Acid (H2SO4): J.T. Baker Instra-analysed grade H2SO4 or equivalent.
4.1.3 50% Sulfuric acid: Dispense 50 mL of reagent grade water into the Teflon storage bottle and slowly add 50 mL of trace pure H2SO4 (sec. 4.1.2).
4.1.4 20% Potassium chloride: Weigh 20 g reagent grade KCl into the Teflon storage bottle and dilute to the 100 mL mark etched on the bottle with reagent grade water.
4.1.5 25% Copper sulfate: Weigh 25 g reagent grade CuSO4 into the Teflon storage bottle and dilute to the 100 mL mark etched on the bottle with reagent grade water. Solution is stable for up to 1 year.
4.1.6 Combined reagent: Combine 100 mL of the Sulfuric acid, 50 mL of the Potassium Chloride, and 100 mL of the Copper Sulfate in a 500 mL Teflon bottle. Solution is stable for up to 1 year.
4.1.7 Nitrogen (N2). Reagent grade N2 that is passed through a gold bead trap attached to the outlet of the tank to remove any Hg.
Three distillation blanks, 3 standards, 10% replicates and 1-2 matrix spikes as appropriate are distilled with every batch of 17 samples to evaluate quality control.
5.1 Distillation blank: A distillation blank is prepared by dispensing approximately 100 mL of reagent grade water into a distillation vial that contains 2.5 mL of the combined reagent. Distillation blanks are used to verify acceptable background levels and calculate daily detection limits during analysis (Analytical Protocol).
5.2 Matrix spike: A matrix spike is prepared by adding a known concentration of working standard to a sample. The matrix spike is used to evaluate accuracy during analysis (Analytical Protocol).
5.3 Sample replicate: A sample replicate is prepared by adding approximately equal amounts of sample to two separate distillation vials. Replicate samples are used to evaluate precision during analysis (Analytical Protocol).
5.4 Standard: A known standard is added to reagent water to evaluate the recovery in comparison to matrix spikes, and to monitor both the condition of the standard and the matrix potential of the reagent water (U.V. treated and untreated).
6.1 Comments: The samples are collected using ultra clean sampling techniques.
6.1.1 Interferences: The distillation procedure is intended to eliminate interferences from the water sample during the ethylation and analysis procedure (Analytical Protocol).
6.1.2 Helpful hints:
6.1.2.1 When working with detection limits in the parts per trillion range, protecting these samples from contamination cannot be over emphasized. The greatest difficulty in low level MeHg analysis is preventing the samples from becoming contaminated. Extreme caution must be used throughout the bottle preparation, collection and distillation procedures to avoid contamination.
6.1.2.2 Maintain a constant temperature on the block during the distillation. As the volume in the distillation vial decreases, a constant temperature setting on the hotplate will result in an elevated temperature at the block.
6.1.2.3 Turn on N2 before making any connections. This will flush the flowmeters and lines with clean N2 and prevent the samples from backing up into the flowmeters. Turn off nitrogen flow only after removing samples.
6.1.2.4 When making the transfer line connection to the receiving vessel, have N2 flowing through the system to assure the system is not blocked or leaking.
6.2 General Description: Samples are added to pre-weighed Teflon stills in approximately 100 mL aliquots. Distillation caps are placed on the vials with the inlet attached to a N2 source and the outlet attached to a pre-weighed Teflon receiving vessel. The distillation vials are heated to approximately 135oC and allowed to distill until a target weight of 85% of the sample has been transferred to the receiver.
6.3 Equipment:
6.3.1 Distillation and receiving vials: 125 mL mercury clean Teflon bottles with 38-430 size screw cap threads.
6.3.2 Transfer caps: The closure of the distillation still has two attached 1/8" lines. Receivers are drilled to accept 1/8” tubing.
6.3.4 Flowmeters: Flowmeters capable of maintaining a flow of 60 mL/min of N2 are placed immediately upstream of the distillation vials to maintain constant and equal flow to all distillation vials. Gas is supplied via 1/8" Teflon tubing.
6.3.5 Distillation block: The distillation block consists of solid aluminum plate drilled to accept 25 stills.
6.3.6 Hot plate: A commercially available hot plate capable of maintaining a distillation block temperature of 135oC.6.3.7 Ice Bath: An ice bath combined with a small volume of ~10 mLs of condensate reagent water introduced into each receiver at setup is sufficient to obtain good condensation of the steam distillate. A cover for the ice bath and tin foil over the stills and lines is required to protect against light catalysed reaction of MeHg
6.3.8 All-plastic pneumatic fixed-volume and variable pipettors in the range of 5 µL to 10 mL.
6.3.9 Analytical balance capable of measuring to the nearest 0.1 mg.
6.3.10 A standard balance capable of measuring to the nearest 0.1 g
6.4 Initial setup and distillation:
6.4.1 The day before a distillation is to be conducted, a batch of samples must be removed from the freezer and allowed to thaw in the dark.
6.4.2 Before beginning sample setup, turn on the hot plate so that it can reach temperature while samples are being prepared.
6.4.3 Pipette 2.5 mL of combined reagent into each distillation vial.
6.4.4 Weigh each vial and record the weight and the unique identification number (engraved on the vial) on the bench sheet.6.4.5 Prepare the three distillation blanks by dispensing approximately 100 mL of reagent grade water into each of three vials. The distillation blanks should be dispersed evenly among the sixteen positions on the distillation block and these positions should be rotated between distillation batches (i.e., distillation batch 1 blanks in position 1,5,10; distillation batch 2 blanks in position 2,6,11; etc.). Record vial i.d., vial plus water weight, and block position on the bench sheet.
6.4.6 Prepare samples identically to blanks using sample water instead of reagent water as above. Be sure to homogenize the sample by inverting the sample bottle several times before dispensing to the distillation vial.6.4.7 The replicate sample is prepared be simply adding similar volumes of the same sample to two separate distillation vials.
6.4.8 The matrix spike is prepared by adding working standard to one of two vials containing similar volumes of the same sample.
6.4.11 Dispense approximately 10 mL of reagent water to each of the receiving vessels. Record jar i.d., vial plus water weight on the bench sheet.
6.4.12 Connect distillation stills to their corresponding receivers and place in the distillation block and ice bath respectively establishing gas flow and noting bubbling in the still and condensate in the receiver.
6.4.13 Adjust the hot plate temperature to maintain a block temperature of 135oC.
6.4.14 Allow the distillation to proceed until the target weight is achieved in the receiving vessel. Check all aspects of the distillation process periodically throughout the distillation. Check for target weight by periodically disconnecting receivers and weighing on the standard balance. Estimate completion times by the rate of distillation.
6.5 Shutdown: As individual distillations reach completion, the distillation vials can be removed from the block by lifting the still, and then disconnecting the gas line. Do not disconnect the gas or stop the flowmeter first.
6.5.1 Thoroughly rinse the cap and transfer line with copious amounts of reagent water. Place the caps in the laminar flow hood to dry.
6.5.2 After the last distillation vial has been removed from the block, the N2 flow and the hot plate should be shut off.
6.5.3 After replacing each drilled jar lid with the numbered solid jar lid, place in the refrigerator until analysis. Ideally this analysis should be done in 3-5 days, and it is our practice to do the analysis the following day.
6.5.4 Wash the distillation vials and receivers in tap water and lab soap and place in the holding bin for the acid cleaning process. Hot water, and where necessary U.V. light or acid can be used to clean still lines and caps if blanks begin to indicate a problem.
7.1 Method source:
Horvat, M., Liang, L., Bloom, N.S. 1993, Comparison of distillation with other current isolation methods for the determination of methyl mercury compounds in low level environmental samples. Part II. Water. Analytica Chimica Acta. 282: 153-168
Olson, M.L. Cleckner, L.B., Hurley, J.P., Krabbenhoft, D.P., Heelan, T.W. 1997, Resolution of matrix effects on analysis of total and methyl mercury in aqueous samples from the Florida Everglades. Fresenius Journal Analytical Chemistry. 358: 392-396
Bloom, N.S. 1989, Canadian Journal of Fisheries and Aquatic Sciences. 46: 1131
7.2 Deviations from source method and rationale:
7.2.1 Deviations from Horvat et al are outlined in Olson et al 1997.