Environ. Sci. Technol. 2008, 42, 6604–6610
Automated Simultaneous Analysis of Monomethyl and Mercuric Hg in Biotic Samples by Hg-Thiourea Complex Liquid Chromatography Following Acidic Thiourea Leaching CHRISTOPHER W. SHADE* Quicksilver Scientific, LLC 1376 Miners Dr., Ste. 101 Lafayette, Colorado 80026
Received January 18, 2008. Revised manuscript received May 6, 2008. Accepted May 14, 2008.
A simple leaching procedure has been validated for quantitative isolation of both monomethyl (CH3Hg+) and inorganic (HgII) mercury from fresh or dried biotic tissue for simultaneous analysis via separation and quantification with Hg-thiourea complex liquid chromatography cold vapor atomic fluorescence spectrometry (HgTU/LC-CVAFS). The leaching solution comprises thiourea, hydrochloric acid, and glacial acetic acid and works by protonating thiol binding sites and forming watersoluble cationic CH3HgSdC(NH2)2+ and Hg[SdC(NH2)2]22+ complexes, which are easily separated from the solid matrix. The isolated complexes are preconcentrated online by either thiol resin trapping or a new iodide-complex polydivinylbenzene resin trapping (I-PDVB). The I-PDVB trapping involves only one reagent addition, requires no pH adjustments, and is quantitative over a large range of volumes and flow rates. The chromatography system can use either ion chromatography or a new ion-pairing reversed phase separation coupled to cold vapor generation and atomic fluorescence detection. The system allows quantitative sample introduction and yields absolute detection limits of 0.4 pg and 0.7 pg, for CH3Hg+ and HgII respectively, enabling relative detection limits as low as 4 and 7 pg g-1 with 100 mg samples, and yields %CV routinely less than 5% with well homogenized samples. Accuracy for both forms of mercury has been validated with multiple biotic reference materials and by comparison of the sum of CH3Hg+ and HgII with total Hg on a variety of different biotic sample types (n ) 49). The system can be calibrated with either aqueous standards or leached reference materials.
Introduction Mercury is well-known for its bioaccumulative nature, and this danger to the human food supply has resulted in numerous studies of bioaccumulation in aquatic food chains (1–5) and recently even terrestrial food chains (6). The focus of such studies has appropriately been the distinctly bioaccumulative form of mercury, monomethylmercury (CH3Hg+); however, inorganic mercury (HgII) is also very toxic (7) and not without potential danger to foodwebs. Though acute toxicity from HgII to wildlife would only likely occur in areas of high mercury loads, the toxicity of HgII is well * Corresponding author phone: +01 (303) 263-6903; e-mail: chris@ quicksilverscientific.com. 6604
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established in toxicology literature and stems from (1) its place as the “softest” of the transition metals (8) and consequent high affinity for biological sulfhydryl groups (9) and (2) its ability to promote oxidative tissue damage (7). Its affinity for soft base groups leads to disruption of the cycling of the redox-buffer glutathione and associated free-radical quenching enzymes, and displacement of native metals centers of enzymes, DNA repair proteins, and “Zn-finger” proteins (10). Recent computational modeling even points to the possibility of displacing Zn2+ from catalytic proteins with more “borderline” anchoring groups such as N and O groups of His and Asp/Glu (11). Mercuric mercury’s propensity to promote oxidation has been clearly documented, especially in relation to its nephrotoxicity, where multiple pathways, including catalytic dismutation of superoxide (12) and depletion of glutathione can result in mitochondrial disruption through elevated mitochondrial hydrogen peroxide generation and consequent lipid peroxidation and membrane depolarization (13). Thus, despite its diminished propensity for bioaccumulation, at any one trophic level HgII has the potential to impair biologic integrity, and in fact clear evidence of toxicity to fish has been demonstrated in studies in Spain (14) and Michigan (15) where distinct oxidative-stress related pathologies of the liver were correlated with elevated tissue HgII levels. Since biomonitoring programs seek to assess threats to the biological integrity of environmental system, biotic HgII could be a revealing measurement. Clearly direct speciation (separation and measurement of both CH3Hg+ and HgII) is the most accurate and informative way to assess impacts to ecosystems from Hg pollution. However, for widespread speciation-based biomonitoring programs, or even just CH3Hg+-based programs such as is proposed in the Environmental Protection Agency’s (EPA) Recommended Water Quality Criteria for Methylmercury, to be economically feasible, a method with simple sample preparation, low detection limits, and rapid and automated analysis is needed. Current methods for speciation analysis require two separate analyses: one for CH3Hg+ (EPA Methods 1630) and one for total Hg-HgT (EPA Method 1631), with HgII being calculated from the difference. EPA Method 1631 now has several automated analyzers supporting it, making it easier to perform than it was a decade ago, but the resultant number for HgII suffers from the combined uncertainty of two measurements and is an added cost. EPA method 1630 uses either alkaline digestion or distillation prior to ethylation and gas chromatographic separations of ethylated analogs of CH3Hg+ and HgII. Distillation suffers from being timeconsuming, having limited and expensive scalability, and only yielding a value for CH3Hg+. Alkaline digestion is scalable as a digestion method and has shown some capacity for measurement of both CH3Hg+ and HgII (16), but the complete dissolution of the matrix yields interferences in the ethylation process that limit the amount of digestate analyzed and thus prohibits a low detection limit. Nitric acid digestion has been shown to extract CH3Hg+ quantitatively and allow larger aliquots for ethylation but it can not quantify HgII and the digestion conditions must be very tightly controlled (17). Some HPLC-ICP-MS methods have employed 2-mercaptoethanol for leaching and simultaneous analysis of CH3Hg+ and HgII (18), but this method lacks a preconcentration method, limiting its ability to analyze zooplankton and insects. Attempts to improve upon the reigning analytical systems have featured creative chemical strategies, but no singular system has been able to surmount all challenges 10.1021/es800187y CCC: $40.75
2008 American Chemical Society
Published on Web 07/24/2008
FIGURE 1. Effect of pH on side reaction coefficient, alpha (a measure of conditional ligand binding strength; for ligands (L),rL ) {[HgII · L]/[Hg2+]}) during (a), leaching and preconcentration, and (b), elution. TU ) thiourea at 0.135 M; R-SH ) generic thiol at 0.1M; I- ) iodide, Ia at 10 mM, Ib at 10 µM (representing an upper limit of [I-] left in the column interstices after postload 1 M HCl rinse). Numbers show conditions for key process points: 1 ) the biotic matrix, 2 ) eluant leaching, 3a ) I-PDVB preconcentration, 3b ) thiol resin preconcentration, 4 ) elution and separation. Thiol modeled with log β ) 40 for 2L:1 M complex; other constants from NIST database (25). necessary to improve analytical conditions in mercury speciation analysis. Hg-thiourea complex liquid chromatography (HgTU/LCCVAFS (formerly Hg-thiourea complex ion chromatography (19) but changed here to reflect addition of ion-pairing reversed phase separation) features a systematic utilization of metals speciation to direct a series of ligand exchange reactions and liquid-solid phase transfers to concentrate, separate, and analyze ionic forms of mercury. The cornerstone of the system is the use of thiourea (TU) as a complexing agent. Thiourea, [(NH2)2CdS]0, is a unique sulfur-based ligand (a thione) that combines the strong complexation properties of thiols (is a strong Lewis base; log K1 for Hg2+ ) 11.34) with the protonation-resistant properties of halides (is a weak Brønsted base; log KH ) -1.34). Thus, the pH scale can be effectively used to establish a predominance of thiol or thiourea complexation (Figure 1), giving extensive flexibility to develop system-compatible applications for different sample types. Since ionic mercury forms coordinate to sulfhydryl (thiol) groups in fish tissue (20) and in natural organic matter in soil (21, 22) and water (22), such samples can be efficiently and effectively leached with acidic thiourea solutions (Figure 1). Thiourea features the additional benefit of being a strong chaotrope (23), a molecule that helps disrupt hydrogen bonding in protein structure, which may allow
better access of the leaching solution to the binding sites, especially those at the hydrophobic core of the protein. The HgTU/LC-CVAFS system features several sample introduction techniques. Samples leached in system eluant can be introduced by a simple sample loop injection when concentrations are high or for screening large numbers of samples. For more precise and low-level measurement two different online solid-phase extractions strategies can be employed: thiol resin preconcentration and hydrophobic resin preconcentration. Shade and Hudson (19) features online thiol-resin preconcentration for sample enrichment and recently Vermillion and Hudson (24) demonstrated use of thiol resin techniques for analysis of CH3Hg+ in humicrich surface and groundwater samples. In this paper, we introduce a new technique based on formation of neutral iodide complexes, CH3HgI and HgI2, and their online enrichment onto a polymeric support, Jordi FLP’s 100% polydivinylbenzene (PDVB) resin. Though thiol enrichment is an excellent technique, the iodide-PDVB (I-PDVB) technique brings many distinct advantages: (1) it involves no pH adjustment and thus is simpler and has less opportunity for error or contamination, (2) the resin involves no pendant attachment of ligands that can be oxidized or defunctionalized over time, and (3) the exchange reaction is a simple partitioning and so will not suffer from kinetic constraints that may affect HgII, which is less kinetically labile than CH3Hg+. The use of I- fits seamlessly with the Hg-TU/LC use of speciation. Iodide is a stronger ligand than TU (Figure 1) and a stronger acid and thus can be added straight to the sample leachate to effect the change in speciation; however, due to the hydrophobicity of the I- complexes, I- can not be used to leach samples as the complexes adsorb strongly to the particles. Thus the two step procedure of first isolating hydrophilic TU complexes by leaching and filtering and then forming hydrophobic complexes prior to preconcentration creates an efficient and elegant system that can facilitate significant advances in understanding of mercury’s role in environmental and human toxicology through high-throughput speciation analysis. The present work uses both data generated for validation studies and data gathered from commercial laboratory use over the course of almost two years to present (1) a simple scheme for quantitative isolation of CH3Hg+ and HgII from biotic materials using acidic-thiourea leaching, (2) a new preconcentration method based on neutral iodide complex formation hydrophobic solid-phase extraction, (3) an ionpairing reversed phase separation scheme, and (4) an improved oxidation step for online cold-vapor generation.
Experimental Section Speciation System. The original analytical system, Hgthiourea complex ion chromatography/cold vapor atomic fluorescence spectrometry (HgTU/IC-CVAFS), is described in detail in Shade and Hudson 2005 (19). Briefly, it consists of ion chromatographic separation of cationic Hg-thiourea complexes, followed by sequential oxidation of CH3Hg+ to HgII, stannous chloride reduction of HgII to Hg0, evaporation of Hg0 into an Ar carrier, drying of the sample gas, and finally atomic fluorescence detection. Prepared samples can be directly injected by sample loop or preconcentrated onto an online trap (either thiol functionalized or new hydrophobic polymer resin traps) fitted to the sample loop of the LC. New changes in chemistry and equipment presented in this work are detailed in Figure 2. Combustion Analyzer. A NIC MA-2000 (Nippon Instrument Corporation, Osaka, Japan) was used for total Hg measurements by combustion (EPA Method 7473). Some modifications to the manufacturer’s design were employed. The system typically uses house air drawn through the system by a vacuum pump at 500 mL min-1 air. The air inlet was VOL. 42, NO. 17, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Analytical system schematic. Components as follows: Autosampler ) Gilson model 222 with controller; HPLC ) Lab Alliance Series III dual-piston all PEEK flowpath high pressure pump (Chromtech); HPP ) high pressure peristaltic pump, Masterflex 7523-30 digital console drive fitted with 77390-00 PTFE tubing pump head (Cole-Parmer); HPIV ) Rheodyne PEEK 6-port high pressure injection valve (Chromtech); PC ) preconcentration column (see Experimental Section); SC ) separation column (see specifications above; PDVB column packed by Column Engineering, resin from Jordi, FLP); CH ) FIAtron TC30 column heater with TC50 controller (30 °C); PP ) Hewlett-Packard 89092 peristaltic pump fitted with minicartridge pump head (Cole-Parmer) and three stop tubing; GLS ) thin-film diffusion gas-liquid separator, custom fabricated borosilicate glass; NDM ) nafion drying membrane, MD-050-24P-2 (Perma-Pure); AFS) atomic fluorescence spectrometer, Tekran 2500 (Tekran Instruments); CS ) chromatography software, Clarity (Data Apex). supplemented with 60 mL min-1 pure O2 to accommodate the low pO2 (lower partial pressures) at the high altitude of our Colorado laboratory. The gold trap furnished by the manufacturer was replaced with a solid-gold bead trap made by Tekran Instruments and designed for continuous emissions monitoring systems. The system was typically calibrated with DOLT-3 reference material. Reagents. System reagents are described in Shade and Hudson 2005 (19). The mobile phase (eluant) of the chromatography system and the leaching solutions were made from thiourea (ACS grade, Acros Organics, ID no. 13891), HCl (trace metal grade, Fisher Scientific, ID no. A508), glacial acetic acid (HOAc-ACS Plus grade, Fisher Scientific, ID no. A38C). Ion-pairing reagent, sodium octane sulfonic acid (ID no. 41636), was from Acros Organics. Sodium hydroxide (ID no. S318), sodium borate (ID no. S248), and sodium citrate (ID no. S279), were all ACS grade from Fisher Scientific. Potassium bromate was 99+% “extra pure” grade from Acros Organics (ID no. 208855000). Stannous chloride was reagent grade (98%) from Sigma-Aldrich (ID no. 208256). Sodium ascorbate, pharmaceutical grade from NOW foods, was added to the borate and citrate buffers to 1% m/v to help preserve resin-anchored thiols and was added to iodide solutions (KI-ACS grade, Acros Organics, ID no. 41826) at a rate of 25% m/v to prevent oxidation of iodide to iodine, which can decompose CH3Hg+. All mixing ratios are presented as g or mL of reagent per 100 mL of final solution. N.B. reagent grades and manufacturers were found to be important for consistent operation of the analytical system; the grades and manufacturers listed were found to work well. To minimize noise in the baseline, most reagents require cleanup steps. Thiourea is prepared in a 5% m/v stock and 6606
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cleaned with thiol resin prior to dilution and acid additions. Potassium bromate is cleaned by heating to 250 °C for 3 hours. Iodide solutions and buffers are also cleaned with thiol resin prior to use to minimize process blanks. The hydroxide solution is cleaned by purging after addition of 1 mL of SnII solution. Safety. Thiourea is toxic and all mercury calibration stocks should be handled with great care due to extreme toxicity. Standards. A 1 µg mL-1 CH3Hg+-Hg standard was prepared from solid CH3HgCl (Sigma-Aldrich) by dissolving in methanol and then diluting into a solution of 0.2% v/v conc. HCl and 0.5% v/v conc. HOAc; the concentration was standardized against a 1 mg mL-1 HgII SPEX Certiprep standard. Certified reference materials, TORT-2 (lobster hepatopancreas), DOLT-3 (dogfish liver), and DORM-2 (dogfish muscle), and LUTS-2 (slurried lobster hepatopancreas) were obtained from NRC Canada; IAEA 2976 (mussel tissue) was obtained from IAEA in Monaco, and BCR 463 (tuna fish muscle) was obtained from RTC (Laramie, Wyoming). Preconcentration Columns. Microcolumns of (1) a proprietary thiol-functionalized hydroxylated polydivinylbenzene-DVB(OH)-SH, and (2) pure polydivinylbenzene (PDVB) resin were packed by Column Engineering (Ontario, CA). The columns were constructed of PEEK-lined stainless steel with aluminum housing and peek fittings (normally sold prepacked with C18 resin as “Quick-Release Cartridge Guard Column System” through Upchurch Scientific). The thiol resin was synthesized in-house from a brominated hydroxylated polydivinylbenzene base resin purchased from Jodi FLP, Waltham, MA; the PDVB (Soxhlet-extracted 5-20 µm, 1000 Å) was obtained from Jordi FLP as well.
TABLE 1. Biotic Reference Material Recovery with Acidic Thiourea Leaching Coupled to Either Thiol Preconcentration/Ion Chromatography Separation or I-PDVB Preconcentration/Ion-Pairing Reversed Phase Chromatography Separationa thiol preconcentration
I-PDVB preconcentration
CRM
CH3Hg+
HgII
sum/ HgT
CH3Hg+
HgII
sum/ HgT
DOLT-3 certified TORT-2 certified DORM-2 certified BCR 463 Certified IAEA 2976 (ng g-1) certified
1.55 ( 0.074 n ) 6 1.59 ( 0.120 0.152 ( 0.013 n ) 6 0.152 ( 0.013 4.57 ( 0.332 n ) 3 4.47 ( 0.320
1.82 ( 0.101
3.37 ( 0.175 3.37 ( 0.14 0.271 ( 0.022 0.27 ( 0.06 4.81 ( 0.379 4.64 ( 26
1.56 ( 0.055 n ) 23 1.59 ( 0.120
1.83 ( 0.075
3.39 ( 0.13 3.37 ( 0.14
4.17 ( 0.022 n ) 2 4.47 ( 0.320 2.75 ( 0.13 n ) 7 2.83 ( 0.15 26.7 ( 0.54 n ) 13 27.8 ( 1.1
0.246 ( 0.011
4.47 ( 0.033 4.64 ( 26 2.80 ( 0.15 2.85 ( 0.16 58.8 ( 3.07 61 ( 3.6
0.119 ( 0.009 0.243 ( 0.047
0.043 ( 0.014 32.1 ( 2.53
a “Sum” ) CH3Hg+ + HgII; uncertainty for “Sum” is sum of CH3Hg+ and HgII uncertainties. All concentrations in µg-Hg g-1-dw, except IAEA 2976 (ng-Hg g-1-dw).
Leaching Procedure. Sample or reference material (typically 2-100 mg dried or 20-250 mg fresh) was weighed into clean 40 mL I-Chem Series 200 borosilicate vials with PTFElined silicone septa, 5-10 mL of leaching solution added, and the mixture heated to 40 °C for 14 h on a dry-block heater (Thermolyne type 16500). The leaching solution typically comprised 0.135 M TU, 1 M HCl, and 2.61 M HOAc (same as the IC system eluant); several variations of these proportions were successfully employed. Leachates were then filtered through prerinsed (with eluant) 0.45 µm (for dried material) or 0.22 µm (for fresh material) PVDF syringe filters and stored in acid-cleaned 8 mL borosilicate vials with PTFElined polypropylene caps. Sample Introduction. Extracted samples could be introduced into the system by any of three autosamplercontrolled methods: sample loop injections, thiol resin preconcentration, or iodide complexation with polydivinylbenzene resin preconcentration (I-PDVB). Sample loop injections consist of sample loop overfilling and a transfer tubing rinse with system eluant between samples. Preconcentration methods involved (1) column preconditioning, (2) sample loading, (3) postload rinsing, and (4) postinjection transfer tubing rinse. For automated I-PDVB trap loading, sample solutions were brought to 10 mM [I-] by addition of 50 µL of cleaned KI stock (1 M KI in 25% m/v sodium ascorbate solution) per 5 mL of sample. The autosampler controlled the peristaltic pump and injection valve to sequentially deliver ∼2 mL of 50% v/v MeOH to precondition the column, 0.5-5.0 mL of sample solution, and then ∼2 mL of 1 M HCl to rinse sample matrix from the column prior to injection. Transfer tubing was rinsed with system eluant between samples to eliminate carry-over. For automated thiol trap loading, sample solution pH was adjusted to pH 3-4 with 0.75 M sodium citrate and, if necessary, 4 M NaOH. Thiol column was preconditioned by neutralizing with 2.5 mL of 0.01 M sodium borate buffer and the postsample and transfer tubing rinse comprised 1% m/v TU and 5% m/v ethylene glycol adjusted to pH 3.5 with HOAc.
Results and Discussion Accuracy and Precision. The leaching method coupled to the HgTU/LC-CVAFS analytical system proved to be highly accurate, as determined by analysis of certified reference materials (Table 1). Precision of replicate measurements is typically 1-3% for repeat injections of aqueous standards. For solid samples, precision depends on degree of homogeneity; for 5 LUTS-2 samples we measured CH3Hg+ as 1.69 ng g-1-ww with a CV of 3.3% and HgII as 17.4 ng g-1-ww with a CV of 2.1%, and for six chopped fish muscle samples we measured CH3Hg+ as 78.2 ng g-1-ww with a CV of 4.8% and
HgII as 7.0 ng g-1-ww with a CV of 13%. The higher CV’s of the fish samples are consistent with the less vigorous homogenization of the fish muscle compared to the highly homogenized thin slurry LUTS-2 sample. Three duplicated samples of freeze-dried and ground zooplankton samples gave CV’s on replicate measurements of 0.3, 1.6, and 1.3% for CH3Hg+ and 1.3, 3.4, and 4.2% for HgII; the tight replication is indicative of thorough homogenization despite the very small sample sizes used (2-6 mg). This higher degree of variability in HgII replicates seems in some cases to be due to a varying degree of breakdown of CH3Hg+ in the source material during harvesting and handling (i.e., fish have high CH3Hg+:HgII so a small degree of breakdown of the CH3Hg+ in the tissue makes a large change in HgII). Absolute detection limits, determined as three times the standard deviation of seven replicate 1 pg injections, were 0.4 pg for CH3Hg+ and 0.7 pg HgII. Actual detection limits depend on sample mass leached and volume of leachate injected, with limits as low as 4 and 7 pg g-1 possible for CH3Hg+ and HgII, respectively, for 100 mg samples. Thiourea in Leaching and Chromatography. To overcome the inherent limitations of complete matrix dissolution, we employed a leaching procedure utilizing acidic TU as the leaching agent. Acetic acid addition was found to be necessary for complete leaching and is thought, based on conversations with Dr. Howard Jordi about the efficient use of his polydivinylbenzene (PDVB) resins (i.e., electron-rich solvents are needed to coordinate to aromatic rings to avoid tailing of peaks during chromatography with PDVB resin due to coordination of N- or S-containing molecules in mercurythiourea complexes), to eliminate secondary reactions between free electron pairs on the thiourea and elements of the biotic matrix, most likely aromatic components of proteins or carbon-carbon double bonds in lipids. The mobile phase of the LC systems conveniently contains all necessary elements and so is used as the leachant as well, providing there is no ion-pairing reagent in it. Leaching recipes were found to be rather insensitive to composition. Leaching experiments with DOLT-3 reference material showed full recovery with [HCl] between 0.12 and 2.4 at 0.135 M TU and 1.74 M HOAc, [TU] between 0.0135 and 0.270 at 1 M HCl and 1.74 M HOAc, and [HOAc] between 0.174 and 3.48 at 0.135 M TU and 1 M HCl (Supporting Information). Leaching without HOAc or other polar organic solvent lowered recoveries to 85-90%. This broad range of efficacy in the leachate recipe is highly desirable and gives flexibility to adapt the procedure to different sample conditions and to more fragile analytes such as ethylmercury. Though 14 h has been adopted for a convenient overnight leach to ensure good diffusion of the TU into the matrix, most finely divided materials are quantitatively leached in VOL. 42, NO. 17, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 3. Time series for leaching two fresh (nondried) materials, LUTS-2 (homogenized nondefatted frozen lobster hepatopancreas) and chopped fish muscle (catfish sample from client). Samples leached as described in experimental section and analyzed by I-PDVB preconcentration and ion-pairing reversed phase chromatography separation.
FIGURE 4. Recovery dependence on sample mass of DOLT-3. Samples were leached in 0.135 M TU, 1.0 M HCl, 1.74 M HOAc for 14 h at 40 °C and analyzed by I-PDVB preconcentration and ion-pairing reversed phase chromatography separation. Open symbols are fluorescence, closed symbols concentration. Solid lines bracket certified range for CH3Hg+ (squares); dashed lines bracket certified range for HgII (circles). a very short time. A time series of leaching LUTS-2 (homogenized nondefatted lobster hepatopancreas) and ground fresh fish muscle showed complete extraction in 2 h (the shortest time tested) and no change values between 2 and 24 h (Figure 3), providing a broad window for leaching time. For dried and powdered material, such as reference materials, we have seen quantitative extraction with as little as 30 min due to the high surface area to volume ratio. Harder, thicker materials like human fingernails and toenails have required up to 12 h for quantitative extraction (manuscript in preparation). Thus 14 h should be sufficient for all environmental biotic matrixes, including chitinous macroinvertebrate shells. Techniques such as ultrasonication and microwave exposure may shorten this time, but have not been explored here; however, since diffusion limits thicker materials, additional energy input other than thermal is not expected to help. Full recovery was obtained for DOLT-3 with sample masses from 10 to 150 mg (the largest mass tested) using 5 mL of leaching solution (Figure 4). For larger masses (e.g., 6608
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whole large insects or small fish), the leaching volume is scaled up. Preconcentration Methods. The I-PDVB preconcentration method gave equivalent results to the thiol trapping method (Table 1). I-PDVB preconcentration is simpler than thiol trapping (one reagent addition with no pH adjustment) and presents less opportunity for error. The I-PDVB method showed quantitative preconcentration from 0.5 to 10 mL of sample solution (the highest volume tested, data not shown). Iodide concentrations from 0.1 to 100 mM all showed quantitative preconcentration from synthetic samples, but 5-10 mM was required for biotic samples. More extensive data on I-PDVB trapping will be presented in upcoming publications on applications to water samples. Ion-Paring Reversed-Phase Chromatography. The new ion-pairing reversed phase separation has some advantages over the ion chromatography separation. The CH3Hg+ peak is more symmetrical and tails less than with the IC system, the CH3Hg+ peak elutes away from the water dip, and the separation times are easily adjusted, even during the run, with the temperature of the column heater (Supporting Information). Additionally, the reversed phase aspect will allow separation of other mono-organo species (monoethyl-, monopropyl-, and monophenylmercury) that would coelute with monomethylmercury using ion-chromatography. These extended speciation applications will likely require larger column than the 2 × 150 mm column used here. Advantages of the IC column are that the sample throughput is slightly faster, it requires no pre-equilibration with ion-pairing reagent, and it is less prone to fouling from sample constituents (PDVB column should be cleaned thoroughly with methanol or acetone every one to two weeks of use or whenever retention times decrease). Cold Vapor Generation Chemistry. The previous use of UV/H2O2 oxidation was changed to KBrO3, which combines with HCl in the eluant to form HOBr (aqueous bromine) for oxidation. Following oxidation, a mixture of Triton-X and NaOH was introduced to change the pH of the reagent stream before addition of SnII, which is soluble at very low and very high pH, but insoluble at circumneutral pH, creating a risk of solid phase formation during mixing of an acidic reagent stream with alkaline SnII. The Triton-X is employed to prevent precipitation of hydrophobic oxidation reaction byproduct. This combination of changes allowed full reduction efficiency with ∼80% less SnII than was previously used, mostly due to a more complete oxidation of TU. Equivalency of slopes of CH3Hg+ and HgII calibrations demonstrates quantitative oxidation of CH3Hg+. We have also successfully used sodium borohydride as a reducing agent (0.05 mM NaBH4 in 1 M NaOH) in place of SnII; however preconcentration must be used for sample introduction due to interactions of matrix constituents in the sample leachate with the borohydride when employing sample loop injection of biotic leachates. Real Samples Sum vs Total. Though certified reference materials are the definitive way to examine accuracy, they are typically defatted and finely ground, eliminating some potential interferences to quantitative recovery. Comparison of speciation (CH3Hg+ + HgII) and HgT by combustion/ amalgamation/CVAA (EPA Method 7473) on a wide variety of fresh (fish muscle, kidney, and liver) and freeze-dried (fish muscle, liver, and gill, and spiders) samples over the course of a year (Figure 5) demonstrates that biotic samples are composed almost entirely of CH3Hg+ and HgII and that the leaching procedure employed yields quantitative recovery. Samples ranged from 1.7 to 96.3% CH3Hg+ with the sum of CH3Hg+ + HgII equaling an average of 98.5% of HgT with a 95% confidence interval of (4.33%. Speciation has been successfully adapted to alkaline cysteine digestions as well. Though direct acidic TU leaching of fresh tissues has the advantage of keeping the majority of
FIGURE 5. Comparison of Sum of CH3Hg+ + HgII by Hg-TU/LC with HgT by combustion (EPA Method 7473) on a variety of fresh and freeze-dried samples (n ) 49) measured over the course of one year. Spider HgT data is from the Cristol laboratory at the College of William and Mary, using a Milestone DMA-80 according to manufacturer’s specifications. Fish tissue samples were leached as described in the Experimental Section and analyzed by thiol preconcentration with ion chromatographic separation; spider samples were leached in 0.135 M TU, 1.0 M HCl, 1.74 M HOAc and analyzed by I-PDVB preconcentration and ion-pairing reversed phase chromatography separation.
functioning of the kidneys and liver (7). Great ranges of CH3Hg+ and HgII can be seen in samples of fish kidney and liver. One fish liver sample supplied by a client contained almost 2.5 µg g-1 HgII, whereas only 1.2 µg g-1 CH3Hg+ was present in the muscle. Recent work by Drevnick et al. (15) examining toxicity and mercury levels in northern pike liver hypothesized that HgII levels in liver have their origin in HgII content of insect prey. Macroinvertebrates (a main fish prey) are a trophic level that has extreme variability in ratios of CH3Hg+:HgII. For example, spiders in Figure 5 had a range in CH3Hg+ (as %HgT) from 1.7 to 86.4%, with HgII levels as high as 50 µg g-1. In this light, speciation of Hg in both insects and fish organs is necessary to understand bioaccumulation potential through trophic levels as well as toxicity to both macroinvertebrates and predators. Future Applications. Thiourea leaching is obviously very effective and robust. Future work will examine the application of thiourea leaching to sediments (including titrations of sediment binding sites) and to other biologic (e.g., blood) and industrial (hydrocarbons and wastewaters) matrices. Iodide-PDVB loading is extremely efficient and simple. Comparisons to thiol trap loading showed identical performance for small volumes (
