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METHOD 3200 

MERCURY SPECIES FRACTIONATION AND QUANTIFICATION BY MICROWAVE 

ASSISTED EXTRACTION, SELECTIVE SOLVENT EXTRACTION 

AND/OR SOLID PHASE EXTRACTION

 SW-846 is not intended to be an analytical training manual. Therefore, method 

procedures are written based on the assumption that they will be performed by analysts who are 

formally trained in at least the basic principles of chemical analysis and in the use of the subject 

technology. 

 In addition, SW-846 methods, with the exception of required method use for the analysis 

of method-defined parameters, are intended to be guidance methods which contain general 

information on how to perform an analytical procedure or technique which a laboratory can use 

as a basic starting point for generating its own detailed standard operating procedure (SOP), 

either for its own general use or for a specific project application. The performance data 

included in this method are for guidance purposes only, and are not intended to be and must not 

be used as absolute QC acceptance criteria for purposes of laboratory accreditation. 

1.0 SCOPE AND APPLICATION 

For a summary of changes in this version from the previous version, please see Appendix 

A at the end of this document. 

 1.1 This method contains a sequential extraction and separation procedure that may 

be used in conjunction with a determinative method to differentiate mercury species that are 

present in soils and sediments. This method provides information on both total mercury and 

various mercury species. 

 1.2 The speciation of a metal, in this case mercury, involves determining the actual 

form of the molecules or ions that are present in the sample. When combined with an 

appropriate determinative method, this procedure is designed to provide varying degrees of 

mercury species information. All metal speciation methods are operationally defined by the 

level of post-extraction processing and the chosen method of analysis. 

 The environmental mobility and toxicity of mercury in a soil profile depend on its 

speciation. Alkyl mercury species such as methylmercury are at least an order of magnitude 

more mobile than inorganic mercury species, and thus are more toxic and more readily 

bioaccumulated. Soluble inorganic mercury species such as mercury chloride are more easily 

transported by natural process than the other inorganic mercury species and serve as the 

substrate for mercury methylation process (Ref. 1). These extractable organomercury species 

and extractable inorganic species contribute the major portion of mercury potential toxicity in the 

soils. The mercury species that fall into the "semi-mobile" category such as elemental mercury 

are less toxic than extractable mercury species. The "non-mobile" mercury species such as 

mercury sulfide are chemically stable in the soil for geologic time periods and thus are least 

toxic. 
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 Examples of the operationally-defined mercury fractions and individual species that may 

be determined using this procedure are presented in the table below. 

Operationally-Defined Mercury Fractions Individual Mercury Species CAS No.a

Total Mercury

Extractable 

Mercury

Extractable Organic 

Mercury

CH3HgCl 

CH3CH2HgCl

115-09-3 

107-27-7 

Extractable Inorganic 

Mercury

HgCl2

Hg(OH)2

Hg(NO3)2

HgSO4

HgO 

Hg2+ complexesc

7487-94-7 

b

10045-94-0 

13766-44-4 

21908-53-2 



Non-extractable 

Mercury

Semi-mobile 

Mercury

Hg0

Hg0

-Md

Hg2+ complexesc

Hg2Cl2 (minor)

7439-97-6 





10112-91-1 

Non-mobile 

Mercury 

Hg2Cl2 (major) 

HgS 

HgSe

10112-91-1 

1344-48-5 

20601-83-6 

aChemical Abstracts Service Registry Number 

bNot registered by the Chemical Abstracts Service 

cCertain inorganic mercury complexes may be present in both the organic and inorganic 

extractable fractions 

dThis represents a mercury-metal amalgam 

 1.3 Quantification of mercury in the different fractions may be performed using any 

suitable technique with appropriate precision and accuracy, for example Method 6800, 7473, 

1631, or Methods 7470 and 7471. Other analytical techniques, such as gas 

chromatography/mass spectrometry (GC/MS), ion chromatography or high performance liquid 

chromatography (HPLC) with either GC/MS or inductively coupled plasma-mass spectrometry 

(ICP-MS) detection (Method 6020), or other hyphenated and/or mass spectrometric techniques, 

may be employed if performance appropriate for the intended application can be demonstrated. 

This method may also be applicable to other matrices, such as industrial and municipal waste 

materials, but its performance on such matrices has not yet been evaluated. Method 6800 

(Elemental and Molecular Speciated Isotope Dilution Mass Spectrometry) may also be 

applicable as a diagnostic and validation tool for quantification of selectively extracted mercury 

species, especially when species transformations occur in the sample preparation or analysis 

procedures. 

 1.4 Prior to employing this method, analysts are advised to consult each method that 

may be employed in the overall analysis (e.g., Method 6800, 7473, 7470, or 7471) and the 

manufacturer’s instructions for additional information on quality control procedures, 

development of QC acceptance criteria, calculations, and general guidance. Analysts also 

should consult the disclaimer statement at the front of the manual and the information in 

Chapter Two for guidance on the intended flexibility in the choice of methods, apparatus, 

materials, reagents, and supplies, and on the responsibilities of the analyst for demonstrating 

that the techniques employed are appropriate for the analytes of interest, in the matrix of 

interest, and at the levels of concern. 

 In addition, analysts and data users are advised that, except where explicitly specified in a 
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regulation, the use of SW-846 methods is not mandatory in response to Federal testing 

requirements. The information contained in this method is provided by EPA as guidance to be 

used by the analyst and the regulated community in making judgments necessary to generate 

results that meet the data quality objectives for the intended application. 

 1.5 This method is restricted to use by, or under supervision of, personnel 

appropriately experienced and trained in the use of metal speciation and analysis techniques. 

Each analyst must demonstrate the ability to generate acceptable results with this method. 

2.0 SUMMARY OF METHOD 

 2.1 For the determination of extractable mercury species, a representative sample 

aliquot is extracted with an appropriate volume of solvent at elevated temperatures. Extraction 

is accomplished with the aid of either microwave irradiation or ultrasound. 

 2.2 Following initial extraction the resultant extracts are separated from the remaining 

sample matrix for analysis of extractable mercury by an appropriate technique. The residual 

sample matrix may be analyzed for non-extractable mercury using an appropriate technique. 

 2.3 The method also has provisions for the separation of the extractable mercury 

fraction into inorganic and organic mercury fractions or individual species. The inorganic and 

organic mercury fractions may be separated by using a solid-phase extraction procedure. 

Individual species may be separated and determined by using an HPLC or other appropriate 

separation device coupled to an appropriate detector. 

 2.4 The method also has provisions for the separation of the non-extractable mercury 

fraction into semi-mobile and non-mobile mercury fractions using sequential acid extraction and 

digestion. 

3.0 DEFINITIONS 

 3.1 Species - The actual form of a molecule or ion that is present in a sample. Ref. 2 

contains additional definitions for “species.” 

 3.2 Sub-speciation - The process by which mercury species in the extractable mercury 

or non-extractable fraction are further subdivided. Ref. 2 contains additional definitions for “subspeciation.” 

 3.3 Total mercury - Total mercury content within the sample, including all inorganic, 

organic, and complex forms of mercury. 

 3.4 Extractable mercury - An operationally-defined fraction of mercury which can be 

extracted from the sample using the protocols described within this method. The extractable 

mercury is meant to represent both organic and inorganic forms of mercury which are more 

labile. 

 3.5 Extractable inorganic mercury -- An operationally-defined subset of the extractable 

mercury species, i.e., the fraction of inorganic mercury species which can be extracted from the 

sample using the protocols described within this method. 

 3.6 Extractable organic mercury - An operationally-defined subset of the extractable 

mercury species, i.e., the fraction of organic mercury species which can be extracted from the 
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sample using the protocols described within this method. 

 3.7 Non-extractable mercury - The operationally-defined fraction of mercury remaining 

in the sample after using the extraction protocols described within this method. The nonextractable mercury is meant to represent the least labile forms of mercury. 

 3.8 Semi-mobile mercury - An operationally-defined subset of the non-extractable 

mercury species, i.e., the fraction of mercury species which can be extracted from the sample 

using the mild-acid extraction protocol(s) described within. 

 3.9 Non-mobile mercury - An operationally-defined subset of the non-extractable 

mercury species, i.e., the fraction of mercury species which can be extracted from the sample 

using the harsh-acid extraction protocol(s) described within. 

 3.10 Refer to Chapter One, Chapter Three, and the manufacturer’s instructions for other 

definitions that may be relevant to this procedure.

4.0 INTERFERENCES 

 4.1 Solvents, reagents, glassware, and other sample processing hardware may yield 

artifacts and/or interferences to sample analysis. All of these materials must be demonstrated 

to be free from interferences under the conditions of the analysis by analyzing method blanks. 

Specific selection of reagents and purification of solvents by distillation in all-glass systems may 

be necessary. Refer to each method to be used for specific guidance on quality control 

procedures and to Chapter Three for general guidance on the cleaning of glassware. 

 4.2 Transformations among mercury species have been reported and experimentally 

verified. For example, methylmercury formed during sample processing from inorganic 

mercury, may cause positive biases in the methylmercury results (Ref. 3 through 5). Also, a 

conversion of methylmercury and ethylmercury to inorganic mercury has been observed under 

certain sample processing conditions (Figures 1 and 2) (Ref. 6). 

 4.3 The possibility of species interconversions cannot be eliminated due to the 

necessary reagents, sample matrix, the combination of reagents and matrix, and/or the 

extraction method used. Method 6800 has successfully been used to monitor and correct for 

such species transformations during speciation of chromium and mercury (Ref. 7-9). 

 4.4 When non-specific detection techniques are employed, the analyst must be aware 

of possible interferences. For example, certain organic compounds may absorb at the 

wavelength of interest when ultraviolet (UV) detection is used. 

NOTE: The adjustment of the pH during certain sample processing steps may result in the 

formation of a precipitate. Filtration (10-μm pore size or less) may be applicable to 

remove such a precipitate. Low recoveries may result from co-precipitation of mercury 

and other sample components. Rinsing the precipitate on the filter with a 0.1% HCl 

solution has been demonstrated to minimize this problem. 

5.0 SAFETY 

 5.1 This method does not address all safety issues associated with its use. The 

laboratory is responsible for maintaining a safe work environment and a current awareness file 

of OSHA regulations regarding the safe handling of the chemicals listed in this method. A 
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reference file of material safety data sheets (MSDSs) should be available to all personnel 

involved in these analyses. 

 5.2 The proper handling of inorganic and organomercury compounds cannot be 

overemphasized. Exposure to organo (alkyl) mercury compounds may cause damage to the 

central nervous system, emotional disturbances, or irritation of the eyes and skin, and may even 

lead to death (Ref. 10, 11). 

 5.3 The use of commercially-available protective gear, such as gloves made of nitrile, 

polyethylene (PE) and ethylvinyl alcohol (EVA) laminate or other appropriate material is 

required. Latex gloves are not suitable for the handling of organomercury compounds (Ref. 11) 

and must not be used. In addition, appropriate eye protection should be used. 

 5.4 If any organomercury compound makes direct contact with the gloves, remove the 

gloves immediately, dispose of them properly, and put on new gloves immediately. These 

materials only provide temporary protection. Consult the glove manufacturer for permeation 

rates and times. If contact should occur with the skin or eyes, flush with large amounts of water 

and seek medical attention immediately. Further information on the safety guidelines on the 

handling of inorganic and organo (alkyl) mercury can be obtained from the material safety data 

sheets for the substances. 

 5.5 The preparation and use of concentrated solutions or samples should be carried 

out in a fume hood (some organomercury compounds are odorless). It is advisable to work 

inside a plastic container with absorbent pads so that accidental spills will be contained. 

 5.6 In the event of a spill, the area should be well ventilated and any ignition sources 

removed. If the spill is a solid, collect and dispose of the material in a sealed container. If the 

spill is a liquid, absorb on paper towels and discard in a designated waste bin. The 

contaminated area can also be cleaned with 0.05% (v/v) 2-mercaptoethanol solution to complex 

and remove the spilled mercury. 

 5.7 The extraction process may generate a moderate amount of pressure. Vessels 

used for extraction should be capable of withstanding these pressures or have pressure relief 

mechanisms or should be vented during the extraction process. 

 5.8 For safety precautions associated with the determinative methods, consult those 

methods directly. 

6.0 EQUIPMENT AND SUPPLIES 

 The mention of trade names or commercial products in this manual is for illustrative 

purposes only, and does not constitute an EPA endorsement or exclusive recommendation for 

use. The products and instrument settings cited in SW-846 methods represent those products 

and settings used during method development or subsequently evaluated by the Agency. 

Glassware, reagents, supplies, equipment, and settings other than those listed in this manual 

may be employed provided that method performance appropriate for the intended application 

has been demonstrated and documented. 

 This section does not list all common laboratory glassware (e.g., beakers and flasks) 

that might be used. 

 6.1 Analytical balance - Accurate to 0.01 g 
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 6.2 Vials/bottles, amber glass - Sizes as appropriate, e.g., 20-mL, with PTFE-lined 

screw-caps or crimp-tops for storage of extracts. 

 6.3 Heating sources - Non-sonicating heating sources equipped with adjustable 

heating control able to maintain a temperature of 95 ± 2 °C (e.g., microwave heat unit, hot block, 

hot water bath or other equivalent) 

 6.4 Sonication heating source - Bath or horn type 

 6.5 Graduated cylinder or equivalent volume measuring device 

 6.6 Volumetric flasks - Sizes as appropriate 

 6.7 pH measuring device - Universal pH paper or calibrated pH meter 

 6.8 Solid-phase extraction system - Visiprep solid-phase extraction manifold (Supelco 

or equivalent system). Consult the manufacturer’s recommendations for the glassware and 

hardware necessary to perform sample extractions. 

 6.9 Solid-phase extraction column - 6-mL glass reaction tubes (Supelco) or equivalent, 

complete with PTFE frits (2 per tube) 

 6.10 Filtration device - Vacuum or manual 

 6.11 Filters - The filter media should have an effective pore size of 1.0 μm or less (glass 

fiber filters are known to work effectively). 

 6.12 Temperature measurement device - Device should be capable of measuring up to 

100 °C accurate to ± 0.1 °C. 

 6.13 Vortex mixer or equivalent 

 6.14 Centrifuge - Maximum speed of at least 3200 rpm and capable of handling 10-mL 

or greater centrifuge tubes 

 6.15 Centrifuge tubes - Disposable glass centrifuge tubes with capacity of at least 10 

mL with snap-on cap 

6.16 Microwave solvent extraction apparatus 

 6.16.1 The temperature performance requirements necessitate that the 

laboratory microwave extraction system be capable of sensing the temperature to within ± 

2.5 oC and automatically adjusting the microwave field output power within 2 sec of 

sensing. Temperature sensors should be accurate to ± 2 oC. Temperature feedback 

control provides the primary performance mechanism for the method. Measurement in at 

least one vessel is required and a rotating turntable to homogenize the microwave field to 

all samples is required for most systems. 

 6.16.2 Microwave extraction vessels are needed. Vessels are available that can 

accommodate 1-g to 10-g samples. In addition the vessel apparatus must accommodate 

the necessary amount of solvent and if appropriate an inner vessel and stir bar and 

secondary microwave energy absorber. Vessels should be essentially transparent to 

microwave energy (with the exception of purposeful microwave absorbing apparatus 

components), relatively inert to reagents and sample components, and capable of 
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withstanding the temperature and pressure requirements (minimum conditions of 200 °C 

and 442 psi) necessary to perform this procedure. Follow the manufacturer’s instructions 

regarding cleaning, handling, and sealing the vessels (Ref. 12). 

7.0 REAGENTS AND STANDARDS 

 Reagent-grade chemicals must be used in all tests. Unless otherwise indicated, it is 

intended that all reagents conform to the specifications of the Committee on Analytical 

Reagents of the American Chemical Society, where such specifications are available. Other 

grades may be used, provided it is first ascertained that the reagent is of sufficiently high purity 

to permit its use without lessening the accuracy of the determination. Reagents should be 

stored in glass to prevent the leaching of contaminants from plastic containers. 

7.1 Hydrochloric acid (concentrated, 12 M), HCl - Certified ACS Plus grade or 

equivalent 

7.2 Sodium hydroxide, NaOH - Certified ACS grade or equivalent 

7.3 Sodium chloride, NaCl - Certified ACS grade or equivalent 

7.4 Copper (II) chloride dihydrate, CuCl2·2H2O - Analytical reagent grade or equivalent 

7.5 Nitric acid (concentrated, 16 M), HNO3 - Certified ACS Plus grade or equivalent 

7.6 Hydrochloric acid (6 M), HCl - Prepared by dilution of 12 M HCl in reagent water 

7.7 Sodium hydroxide (10 M), NaOH - Prepare by dissolution of solid NaOH pellets of 

appropriate purity in reagent water 

7.8 Ethanol, CH3CH2OH - HPLC grade or equivalent 

7.9 Silver nitrate, AgNO3 - Certified ACS grade or equivalent 

 7.10 HPLC subspecies option mobile phase components 

 7.10.1 Methanol, CH3OH - HPLC grade or equivalent 

 7.10.2 2-Mercaptoethanol, HSCH2CH2OH - reagent grade 

 7.10.3 Ammonium acetate, NH4CO2CH3 - reagent grade 

7.11 Acetic acid, CH3CO2H - HPLC grade or equivalent 

 7.12 Sulphydryl cotton fiber (SCF) separation and concentration option 

 7.12.1 Materials for preparing SCF 

 7.12.1.1 Cotton - Mercury-free cotton fiber 

 7.12.1.2 Mercaptoacetic acid (97+%), HSCH2CO2H - analytical reagent 

grade 

 7.12.1.3 Acetic anhydride, (CH3CO)2O - HPLC grade or equivalent 
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 7.12.1.4 Sulfuric acid, (concentrated, 18 M), H2SO4 - analytical reagent 

grade or spectrograde quality 

 7.12.2 SCF eluent solutions 

 7.12.2.1 SCF eluent 1, an aqueous solution containing 1.0 M HCl and 

1.0 M NaCl -- Prepare by diluting 20.7 mL of concentrated HCl to 250 mL in 

reagent water, then dissolving 14.6 g of NaCl in the prepared 1.0 M HCl. 

 7.12.2.2 SCF eluent 2, an aqueous solution containing 6 M HCl, 

saturated NaCl, and 0.1% CuCl2⋅2H2O -- Prepare by diluting 124 mL of 

concentrated HCl to 250 mL in water, then adding 0.25 g of CuCl2⋅2H2O and 11.0 g 

of NaCl. 

NOTE: A portion of the solid NaCl may remain un-dissolved and settle to the 

bottom of the vessel. The top portion of this solution should be used. 

 7.13 Reagent water - Reagent water must be interference free. All references to water 

in the method refer to reagent water unless otherwise specified. 

 7.14 2.0% (v/v) HCl + 10% (v/v) ethanol extraction solution - Prepared by dilution of the 

proper amount of concentrated HCl and ethanol in reagent water. 

 7.15 1:2 (v/v) HNO3 extraction solution - Prepare by combining 1 part concentrated 

HNO3 with 2 parts reagent water. 

 7.16 1:6:7 (v/v/v) HCl : HNO3 : reagent water - Prepare by combining 1 part 

concentrated HCl, 6 parts concentrated HNO3 with 7 parts reagent water 

 7.17 Chloride ion test solution - 0.1 M silver nitrate in 0.1 M HNO3. Commercially 

prepared 0.1 M silver nitrate solution is available. 

 7.18 4 M HNO3 extraction solution -

 Prepared by dilution of the proper amount of 

concentrated HNO3 in reagent water.

 7.19 Inorganic mercury standard solutions - Commercially-prepared standards 

containing natural isotopically-abundant inorganic mercury species and isotopically-enriched 

inorganic mercury species, such as Hg2+, are available (Ref. 13, 14). Standard solutions can 

also be prepared by dissolution of the selected pure solid mercury compound in an appropriate 

solvent. Mercury compounds must be of known concentration. All solvents used for standards 

preparation must be analytical reagent grade or equivalent. 

 7.20 Organic mercury standard solutions - Commercially prepared standards containing 

natural isotopically abundant CH3HgCl or other organic mercury species and/or isotopicallyenriched CH3HgCl or other organic mercury species are available (Ref. 13, 14). Standard 

solutions can also be prepared using the pure organic mercury compound and appropriate 

solvents (Ref. 4). Pure compounds must be of known concentration. Solvents used for 

standards preparation must be analytical reagent grade or equivalent. 

 7.21 Sodium acetate, anhydrous, NaCH3CO2 - Certified ACS grade or equivalent 

 7.22 Sodium acetate trihydrate, NaCH3CO2·3H2O - Certified ACS grade or equivalent 
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 October 2012 

 7.23 0.2 M acetate buffer solution (pH 3.0) - Mix 11.4 mL of acetic acid and 0.2789 g of 

anhydrous sodium acetate or 0.4627 g of sodium acetate trihydrate in reagent 

water. Dilute to 1 L with reagent water. Measure the pH of the buffer solution and 

if needed adjust the pH with a strong acid or base.

8.0 SAMPLE COLLECTION, PRESERVATION, AND STORAGE 

 Sample collection, preservation and storage requirements may vary by EPA program and 

may be specified in a regulation or project planning document that requires compliance 

monitoring for a given contaminant. Where such requirements are specified in the regulation, 

follow those requirements. In the absence of specific regulatory requirements, use the following 

information as guidance in determining the sample collection, preservation and storage 

requirements. 

 8.1 See the introductory material of Chapter Three, “Inorganic Analytes.” 

 8.2 Samples should be collected and placed in containers that are made of glass or 

other appropriate material. 

 8.3 Sample extracts should be analyzed within 5 days (Ref. 15). This unique holding 

time recommendation will be added to the Update V version of Chapter Three. 

9.0 QUALITY CONTROL 

 9.1 Refer to Chapter One for guidance on quality assurance (QA) and quality control 

(QC) protocols. When inconsistencies exist between QC guidelines, method-specific QC 

criteria take precedence over both technique-specific criteria and those criteria given in Chapter 

One, and technique-specific QC criteria take precedence over the criteria in Chapter One. Any 

effort involving the collection of analytical data should include development of a structured and 

systematic planning document, such as a Quality Assurance Project Plan (QAPP) or a Sampling 

and Analysis Plan (SAP), which translates project objectives and specifications into directions 

for those that will implement the project and assess the results. Each laboratory should 

maintain a formal quality assurance program. The laboratory should also maintain records to 

document the quality of the data generated. All data sheets and quality control data should be 

maintained for reference or inspection. 

 9.2 Initial demonstration of proficiency 

 Each laboratory must demonstrate initial proficiency with each sample preparation and 

determinative method combination it utilizes by generating data of acceptable accuracy and 

precision for target analytes in a clean reference matrix. This will include a combination of the 

sample extraction method and the determinative method (e.g., a 7000 series method). The 

laboratory must also repeat the demonstration of proficiency whenever new staff members are 

trained or significant changes in instrumentation are made. See Method 8000 for information on 

how to accomplish a demonstration of proficiency. 

 9.3 Initially, before processing any samples, the analyst should demonstrate that all 

parts of the equipment in contact with the sample and reagents are interference-free. This is 

accomplished through the analysis of a method blank. As a continuing check, each time 

samples are extracted, cleaned up, and analyzed, and when there is a change in reagents, a 

method blank should be prepared and analyzed for the analytical species of interest as a 

safeguard against chronic laboratory contamination. The blanks should be carried through all 
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stages of sample preparation and analysis. When new reagents or chemicals are received, the 

laboratory should monitor the preparation and/or analysis blanks associated with samples for 

any signs of contamination. It is not necessary to test every new batch of reagents or chemicals 

prior to sample preparation if the source shows no prior problems. However, if reagents are 

changed during a preparation batch, separate blanks need to be prepared for each set of 

reagents. 

 The laboratory should not subtract the results of the method blank from those of any 

associated samples. Such “blank subtraction” may lead to negative sample results. If the 

method blank results do not meet the project-specific acceptance criteria and reanalysis is not 

practical, then the data user should be provided with the sample results, the method blank 

results, and a discussion of the corrective actions undertaken by the laboratory. 

 9.4 Sample quality control for preparation and analysis 

 The laboratory must also have procedures for documenting the effect of the matrix on 

method performance (precision, accuracy, method sensitivity). At a minimum, this should 

include the analysis of QC samples including a method blank, a duplicate, a matrix spike and a 

laboratory control sample (LCS) in each analytical batch. Any method blanks, matrix spike 

samples, and replicate samples should be subjected to the same analytical procedures (Secs. 

4.0 and 11.0) as those used on actual samples. An example of Quality Control is described in 

section 13.8 (ref 26), demonstrates a mass balance method for mercury species where the 

interchanges between mercury species are quantified by measuring each species and then 

correcting for the inter-species transformations with method 6800 and obtaining mass balance 

by thermally grouping remaining non-extractable species as a single mercury species. These 

species are added together and compared with total mercury values independently measured 

by methods 3200 and 6800. The species added together equal the total mercury to, within the 

confidence interval, total combined measurement uncertainty to achieve mass balance. Method 

3200 should not be used with other analytical methods except 6800 if the mercury species are 

active and transform from one species to another as demonstrated in Reference 26 and Method 

6800. Species spike recovery is also a method of demonstration of Species stability.

 9.4.1 Method blank - For each analytical batch of samples processed, at least 

one method blank should be carried throughout the entire sample preparation and 

analytical process. A method blank is prepared using the same reagents and quantities 

used with samples and processed through the appropriate steps of the procedure with the 

samples. These steps may include, but are not limited to extraction, chromatographic 

separation, concentration, dilution, filtering, and analysis. If the method blank does not 

contain target analytes at a level that exceeds the project-specific criteria requirements, 

then the method blank would be considered acceptable. In the absence of project-specific 

criteria, if the blank is less than the lower limit of quantitation, less than 10% of the 

regulatory limit, or less than 10% of the lowest sample concentration, whichever is greater, 

then the method blank is considered acceptable. If the method blank cannot be 

considered acceptable, the method blank should be re-analyzed at once, and if still 

unacceptable, then all samples after the last acceptable method blank must be reprepared and re-analyzed along with the other appropriate batch of QC samples. These 

blanks will be useful in determining if samples are being contaminated. If the method 

blank exceeds the criteria, but the samples are all either below the reporting level or below 

the applicable action level or other criteria, then the data should not be rejected based on 

this analysis. 

 9.4.2 Matrix spike (MS)/ Duplicate - For each batch of solid phase samples 

extracted using this method, at least one matrix spike (MS) and one duplicate unspiked 

sample or one matrix spike/matrix spike duplicate (MS/MSD) pair should be carried 
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through the entire sample extraction, preparation and analytical process. An MS is an 

intra-laboratory split sample spiked with a known concentration of each analytical species 

of interest (see Secs. 7.19 and 7.20). Spiking is performed directly on the solid sample 

matrix prior to extraction in order to assess the efficiency of the extraction procedure. The 

decision on whether to prepare and analyze duplicate samples or a MS/MSD must be 

based on knowledge of the samples in the sample batch. If samples are expected to 

contain the target analyte, laboratories may use a matrix spike (MS) and a duplicate 

analysis of an unspiked field sample. If samples are not expected to contain the target 

analyte, the laboratories should use a MS/MSD pair. A separate spiked sample and a 

separate duplicate unspiked sample may be analyzed in lieu of MS/MSD analyses. MS 

samples should be spiked at the project-specific action level or, when lacking projectspecific action levels, between the low- and mid-level calibration standards. Acceptance 

criteria should be set at a laboratory-derived limit developed through the use of historical 

analyses per matrix type analyzed. In the absence of historical data, the accuracy limit 

should be set at ±25% of the spiked concentration for the MS and 25% RPD for the 

MS/MSD or duplicate precision. Matrix spike/duplicate or MS/MSD samples should be 

included whenever a new matrix type is being analyzed. 

 

 9.4.3 LCS - A laboratory control sample (LCS) should be included with each 

analytical batch of samples processed. The LCS consists of an aliquot of a clean (control) 

matrix similar to the sample matrix and of the same weight or volume. The LCS is spiked 

with the same analytes, at the same concentrations, as the matrix spike, when 

appropriate. When the results of the matrix spike analysis indicate a potential problem 

due to the sample matrix itself, the LCS results may be used to verify that the laboratory 

can perform the analysis in a clean matrix. 

NOTE: A standard reference material (SRM), certified reference material (CRM) or 

reference material (RM) may be used in place of an LCS. The SRM, CRM or RM 

generally consists of a commercially-prepared, well characterized matrix similar to 

the sample matrix and containing the analytical species of interest at established 

reference concentration levels. 

 9.5 It is recommended that the laboratory adopt additional quality assurance 

practices for use with this method. The specific practices that are most productive depend upon 

the needs of the laboratory and the nature of the samples. Whenever possible, the laboratory 

should analyze standard reference materials and participate in relevant performance evaluation 

studies. 

10.0 CALIBRATION AND STANDARDIZATION 

 The laboratory microwave system should be calibrated to measure within ±2 °C. Consult 

the manufacturer’s instructions for microwave system calibration. 

11.0 PROCEDURE 

 11.1 The specific procedures that are applied to the sample depend on the extent of the 

mercury speciation information that is required. 

 11.1.1 If only total mercury data are required, then analyze the sample using 

Method 7473 or other suitable procedures, or sum the results for the extractable and nonextractable mercury fractions from Sec. 11.8. 
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 11.1.2 If extractable mercury data are required, then proceed to Sec. 11.2 for the 

mercury extraction procedures and use either the microwave-assisted extraction 

procedure (Sec. 11.2.1) or the ultrasound-assisted extraction procedure (Sec. 11.2.2). 

 11.1.3 If non-extractable mercury data are required, then proceed to Sec. 11.2 

for the mercury extraction procedures, but analyze the residual solids from the extraction 

procedure as stated in Sec. 11.2.1.5 or 11.2.2.8. 

 11.1.4 If data are required for extractable organic mercury, extractable inorganic 

mercury, or both fractions, proceed to Sec. 11.2 for the extraction procedures and then to 

Sec. 11.4 for the separation and concentration of the extractable mercury by solid-phase 

extraction, followed by Sec. 11.5 for the sub-speciation of the extractable organic and 

extractable inorganic mercury fractions. 

 11.1.5 If data for individual extractable mercury species are required, proceed to 

Sec. 11.2 for the extraction procedures and then proceed to Sec. 11.6 for the subspeciation of the individual extractable mercury species by HPLC or appropriate 

hyphenated technique (see Sec. 11.8). 

 11.1.6 If data are required for semi-mobile mercury, non-mobile mercury, or both 

fractions, proceed to Sec. 11.2 for the mercury extraction procedures. Then, process the 

residual solids generated in Sec. 11.2 through the separation and extraction procedures in 

Sec. 11.7 for the sub-speciation of the semi-mobile and non-mobile mercury. 

 11.1.7 For some projects, it may be appropriate or required to report the results 

of the mercury speciation analyses relative to the dry weight of the original sample. The 

calculation of the dry weight of the sample is described in Sec. 12.1. 

 11.2 Extractable mercury 

 11.2.1 Microwave-assisted extraction 

 This extraction involves the use of a solution of 4.0 M HNO3 to extract mercury 

species from soil or sediment samples (Ref. 16-18).

 11.2.1.1 Weigh approximately 1 g (± 0.2 grams, to at least 3 significant 

figures) of homogenized soil or sediment sample into microwave extraction 

vessels. 

 11.2.1.2 Add 10.0 mL of 4.0 M HNO3 to each sample. Add a magnetic 

stirring bar to each vessel for thorough mixing of solvent with the sample. 

 11.2.1.3 Seal microwave vessels and irradiate at 100 °C for 10 min with 

magnetic stirring on. A 2-min ramping time should be used to reach the desired 

temperature of 100 °C. 

 NOTE: Longer ramping time may be used depending on the microwave power 

loading of the system. The extraction time of 10 min should be 

maintained independently of the ramping time. 

 11.2.1.4 Let the vessels cool for safe handling, and then filter the 

extracts through a 0.22-μm glass fiber filter. Store extracts in the cold at 4 °C until 

analysis (within 5 days). This extract contains the extractable mercury species and 

can be analyzed directly, concentrated and then analyzed or divided for further 
SW-846 Update V 3200 - 13 Revision 1 

 October 2012 

processing. See Sec. 11.8 for analysis, Sec. 11.4 for separation and concentration 

and Sec. 11.5 for further speciation. 

 11.2.1.5 The residual solids may be saved for the direct analysis of the 

non-extractable mercury by Method 7473 or may be further extracted as semimobile and non-mobile mercury species. See Sec. 11.8 for analysis and Sec. 11.3 

and 11.7 for further speciation. 

11.2.2 Ultrasound-assisted extraction 

 This extraction involves the use of a solution of 2.0% HCl + 10% ethanol to extract 

mercury species from solid or sediment samples. 

 11.2.2.1 Weigh approximately 1 g (± 0.5 g, to at least 3 significant 

figures) of sample into a centrifuge tube. 

 11.2.2.2 Add 2.5 mL of 2.0% HCl + 10% ethanol extraction solvent to 

each sample. Make sure each tube is properly sealed. 

 11.2.2.3 Vortex the samples for 1 min then centrifuge the samples for 1 

min. Test the pH value of the supernatant. If the pH is greater than 3, add 

concentrated HCl drop-wise; then vortex, centrifuge, and again test the pH. 

Repeat this step until the pH value of the supernatant is between 1.5 and 3. It is 

recommended to add a few drops (1 drop = 0.05 mL; not more than 5 drops) of 

concentrated acetate buffer (0.2 M, pH 3.0) to the supernatant, before adding HCl, 

to achieve this pH with minimal reagent addition. 

 11.2.2.4 Vortex the samples for 1 min. Sonicate samples for 7 min at 

60 ± 2 °C. 

 11.2.2.5 Centrifuge the samples for 5 min at 3100 ± 100 rpm. Decant 

the supernatant into an appropriate container. 

 11.2.2.6 Repeat the extraction (Secs. 11.2.2.2, 11.2.2.4 and 11.2.2.5) 

three more times. Additional pH adjustment (Sec. 11.2.2.3) is not necessary. 

Combine all collected extraction supernatants into the same vessel. 

 11.2.2.7 Add 2.5 mL of reagent water to the sample residue. Vortex 

the samples for 1 min, and then centrifuge the samples for 1 min. Combine the 

water rinse with extraction supernatants from 11.2.2.6. This combined solution 

contains the extractable mercury species and can be analyzed directly, 

concentrated and then analyzed, or divided for further processing. See Sec. 11.8 

for analysis, Sec. 11.4 for separation and concentration and Sec. 11.5 for further 

speciation. 

 11.2.2.8 The residual solids may be saved for the direct analysis of the 

non-extractable mercury by Method 7473 or further extracted as semi-mobile 

mercury species and non-mobile mercury species. See Sec. 11.8 for analysis and 

Secs. 11.3 and 11.7 for further speciation. 

 11.3 Further speciation of extractable mercury and non-extractable mercury 

 Determine if the extractable mercury fraction is to be divided into organic and inorganic 

fractions or into individual mercury species. To concentrate the extractable mercury fraction 
SW-846 Update V 3200 - 14 Revision 1 

 October 2012 

and/or separate it into inorganic and organic mercury fractions, proceed to Sec. 11.4. To 

separate the extractable mercury fraction into individual mercury species, proceed to Sec. 11.6. 

To separate the non-extractable mercury fraction into semi-mobile and non-mobile mercury 

fractions, proceed to Sec. 11.7. 

 Sub-speciation by alternative techniques, such as GC-MS or chelation resins, may also be 

appropriate, provided that adequate performance can be demonstrated. 

 11.4 Isolation of extractable mercury by solid-phase extraction 

 This procedure is appropriate for the separation and concentration of the extractable 

mercury fraction using solid-phase extraction. Adequate separation has been achieved using 

SCF as the solid-phase extraction medium (Ref. 6, 15, 16, 19-21). The medium is commercially 

available (Ref. 13) or can be prepared in house following the procedure described below. 

 11.4.1 Prepare a mixture containing 50 mL of mercaptoacetic acid, 35 mL of 

acetic anhydride, 16 mL of acetic acid, 0.15 mL of sulfuric acid and 5 mL of reagent water 

in a clean vessel. Immerse a 15-g portion of cotton fiber in this mixture. Cover the vessel 

and place it in a constant temperature apparatus capable of maintaining a temperature of 

40 ± 2 °C for four days. Remove the cotton fiber from the reagent mixture and place it in a 

filter funnel (or alternative vacuum filtration device) and rinse with reagent water until the 

pH of the washings is neutral. Dry the cotton fiber (now "SCF") at 40 ± 2 °C for two days. 

Store the SCF in a cold location at ≤ 6 °C or in a refrigerator in a dark bottle wrapped with 

aluminum foil. The SCF product is stable for at least 3-4 months (Ref. 20). Alternatively, 

purchase SCF solid phase columns commercially. 

 11.4.2 Place a PTFE frit at the bottom of an appropriate size column. Add a 

0.2-g portion of the SCF, along with 3 mL of water. Place a second PTFE frit on the top of 

the SCF. Apply pressure to the top frit to compact the SCF into a homogeneous disk 

between the two frits. 

 NOTE: The bed volume of the SCF disk is approximately 0.7 mL per 0.1 gram of SCF 

solid-phase material. For example, in a column with an internal diameter of 

1.1 cm, the height of the disk is about 1.4 cm. Higher capacity can be achieved 

by using more SCF; however the reagents for separation and fractionation should 

be proportionally increased. 

 NOTE: The column should be packed on the same day that the separation procedure will 

be performed and the SCF disks should be prepared with care to avoid 

channeling. If using commercially available SCF disks, then same day packing is 

not required. 

 11.4.3 Just prior to use condition the SCF disk by passing 10 mL of reagent 

water, then 10 mL of 6 M HCl and finally 15 mL of reagent water sequentially through the 

medium using a flow rate of 1 mL/min. 

 NOTE: The conditioning procedure should be performed just before the separation. After 

conditioning, the SCF media must be used within 30 min. 

 11.4.4 Adjust the pH of the extract from Sec. 11.2.1.4 or Sec. 11.2.2.7 of the 

extraction procedure to pH = 6 ± 1 with 10 M NaOH. Filter the solution to retain the 

particles whose sizes are larger than 1.0 μm. Rinse the retained particles with 3 mL 0.1% 

HCl. Combine the filtered solution with the rinse solution. 
SW-846 Update V 3200 - 15 Revision 1 

 October 2012 

 NOTE: The presence of large amount of Fe3+ in the extracted solution will degrade the 

retention and separation ability of the SCF media. It may also induce the 

transformation from alkyl mercury to inorganic mercury under acidic condition. 

After adjusting the pH to 6 ± 1, the precipitation of Fe(OH)3 is achieved. 1.0-μm 

filtration will minimize the presence of Fe3+ in the solution before it is passed 

through the SCF column. The 0.1% (v/v) HCl rinse will minimize the loss of Hg 

species of interest during the filtration procedure (Ref. 6). 

 NOTE: 10 M NaOH is preferred over concentrated NH4OH for pH adjustment because 

NH4OH may precipitate out Hg2+

. 

 11.4.5 Adjust the pH of the filtered solution to pH = 3 ± 1 with 6 M HCl and pass 

through the SCF column using a flow rate of ≤1 mL/min. 

 11.4.6 If only the extractable mercury fraction is of interest, remove the SCF disk 

from the column and analyze it directly using an appropriate determinative method such as 

Method 7473 (Ref. 6, 22) to determine the amount of extractable mercury. 

 11.4.7 Alternatively, the concentrated extractable mercury fraction can be further 

separated into extractable inorganic mercury and extractable organic mercury fractions. If 

such separation is needed, proceed to 11.5. 

11.5 Sub-speciation of inorganic and organic extractable mercury 

 11.5.1 Elute the organic mercury species from the SCF solid phase extraction 

medium by passing 8 mL of eluent 1 (see Sec. 7.12.2.1) through the SCF column at a 

flow rate of ≤1 mL/min, followed by 2 mL of water. This eluent is analyzed using an 

appropriate determinative method to determine the amount of organomercury that has 

been extracted from the sample. See Sec. 11.8 for details. 

 11.5.2 The extractable inorganic mercury fraction will remain on the SCF and 

may be analyzed by an appropriate mercury detection method, either by direct analysis 

of the solid SCF medium or by appropriate dissolution of the medium and subsequent 

analysis. See Sec. 11.8 for details. 

 11.5.3 As another option, the remaining inorganic mercury fraction can also be 

eluted from the SCF medium by passing 8 mL of eluent 2 (see Sec. 7.12.2.2) through 

the SCF column at a flow rate of ≤1 mL/min, followed by 2 mL of water. This eluent is 

analyzed using an appropriate determinative method to determine the amount of 

inorganic mercury that has been extracted from the sample. See Sec. 11.8 for details. 

 11.6 Sub-speciation of extractable mercury by HPLC 

 This procedure is appropriate for the separation of the extractable mercury fraction into 

individual mercury species: HgCl2, Hg(OH)2, Hg(NO3)2, HgSO4, HgO, Hg2+ complexes as Hg2+

ion, and CH3Hg+

 and CH3CH2Hg+ ions. 

 11.6.1 Adequate separation has been achieved by HPLC using a 30 cm x 4.0 

mm, 5-μm pore size C-18 column and a mobile phase containing 30:70 methanol:H2O, 

0.005% 2-mercaptoethanol, 0.6 M ammonium acetate (Ref. 8, 9). 

 11.6.2 To ensure compatibility with the HPLC column, adjust the pH of the 

extract from either Sec. 11.2.1.4 or Sec. 11.2.2.7 or Sec. 11.5.1 or Sec. 11.5.3 to a value 

in the range of 3 to 7. Dilute samples to an appropriate volume using reagent water. 
SW-846 Update V 3200 - 16 Revision 1 

 October 2012 

 NOTE: Certain metals which may be extracted from the soil along with the mercury 

species may precipitate when the pH is raised. 

 11.6.3 A precipitate formation due to the pH adjustment step may interfere with 

the separation or the detection method, in which case it may be necessary to filter the 

sample. Refer to Sec. 11.4.4 

 11.6.4 If the presence of a large amount of inorganic mercury interferes or 

masks the organic mercury peak then the SCF separation described in Sec. 11.4 can be 

used to eliminate this problem. 

 11.6.5 Refer to Method 8000 for additional information on HPLC analysis, 

Method 6800 for speciated isotope dilution, and other appropriate analytical detection 

methods. 

 11.7 Sub-speciation of non-extractable mercury 

 If needed, the remaining matrix material from Sec. 11.2.1.5 or Sec. 11.2.2.8 may 

be further divided as semi-mobile and non-mobile mercury fractions. 

 Sub-speciation of non-extractable mercury by alternative techniques, such as 

Method 3052 or other acid leaching and digestion procedure (Ref. 12, 23-25), may also be 

appropriate. 

 11.7.1 Semi-mobile mercury species 

 11.7.1.1 Add 5 mL reagent water to the remaining sample portion from 

Sec. 11.2.1.5 or Sec. 11.2.2.8. Vortex the sample continuously for 1 min. 

 11.7.1.2 Sonicate the sample for 1 min at 60 ± 2 °C. 

 11.7.1.3 Centrifuge the mixture for 5 min at 3200 rpm. Transfer the 

supernatant to a container. 

 11.7.1.4 Test the supernatant for chloride ions by adding 2-3 drops of 

chloride ion test solution (Sec. 7.17). Formation of a white precipitate or white 

turbidity indicates the presence of chloride ions. 

 11.7.1.5 Repeat the steps in Secs. 11.7.1.1-11.7.1.4 until the decanted 

supernatant is free of chloride ions. After testing for the presence of the chloride 

ion the supernatant should be discarded. 

NOTE: The presence of chloride ions in the soil sample portion may result in the 

extraction of both semi-mobile and non-mobile mercury species in the 

same extraction step. 

 11.7.1.6 Add 5.0 mL of 1:2 (v/v) HNO3 extraction solution to the 

remaining soil sample portion from 11.7.1.3. Make sure each vessel is properly 

sealed. Vortex the sample for 1 min. 

 11.7.1.7 Heat the mixture for 20 min at 95 ± 2 °C in a heating device 

described in Sec. 6.3. 
SW-846 Update V 3200 - 17 Revision 1 

 October 2012 

NOTE: Gas may evolve, therefore, the vessel should be capped loosely during 

heating, unless the vessel is pressurizable. 

 11.7.1.8 Centrifuge the extracted mixture for 5 min. Transfer the 

supernatant to an appropriate container. 

 11.7.1.9 Repeat the steps in Secs. 11.7.1.6-11.7.1.8 once more. 

Combine all collected extraction supernatants in the same vessel. 

 11.7.1.10 Add 5.0 mL of reagent water to the remaining soil sample 

portion. Vortex the sample for 1 min and then centrifuge for 5 min. Decant and 

combine the rinse water with the extract supernatants. This solution contains the 

semi-mobile mercury species and can be analyzed by an appropriate mercury 

detection method. See Sec. 11.8 for details. 

 11.7.1.11 The residual solids may be saved for the analysis of the nonmobile mercury species. 

 11.7.2 Non-mobile mercury species 

11.7.2.1 Analyze the solids from 11.7.1.11 directly for non-mobile 

mercury species (Sec. 11.8). 

11.7.2.2 Alternatively, perform the following extraction (Secs. 11.7.2.3-

11.7.2.7) and analyze the extract by appropriate means (Sec. 

11.8). 

 11.7.2.3 Add 5.0 mL of 1:6:7 (v/v) HCl:HNO3:reagent water extraction 

solution (Sec. 7.11) to the residual soil sample portion from 11.7.1.11. Make sure 

each vessel is properly sealed. Vortex the sample for 1 min. 

 11.7.2.4 Heat the mixture for 20 min at 95 ± 2 °C in a heating device 

described in Sec. 6.3. 

 NOTE: Gas may evolve, therefore, the vessel should be capped loosely during 

the heating, unless the vessel is pressurizable. 

 11.7.2.5 Centrifuge the extracted mixture for 5 min. Transfer the 

supernatant to an appropriate container. 

 11.7.2.6 Repeat the steps in Secs. 11.7.2.3-11.7.2.5 once more. 

Combine all collected extraction supernatants into the same vessel. 

 11.7.2.7 Add 5.0 mL of reagent water to the remaining soil sample 

portion. Vortex the sample for 1 min and then centrifuge for 5 min. Decant and 

combine the rinse water with the extract supernatants from Sec. 11.7.2.6. This 

solution contains the non-mobile mercury species and can be analyzed by an 

appropriate mercury detection method. See Sec. 11.8 for details. 
SW-846 Update V 3200 - 18 Revision 1 

 October 2012 

 11.8 Analysis of specific mercury fractions 

 Many mercury detection techniques are appropriate detection techniques, such as 

Methods 7473, 1631, 7470, 7471, 6020 and 6800. Other mercury detection techniques 

may also be appropriate, provided that adequate performance can be demonstrated. 

 11.8.1 Extractable mercury -- Analyze the extractable mercury fraction from Sec. 

11.2.1.4, 11.2.2.7 or 11.5.3, using an appropriate detection technique. 

 NOTE: Pre-concentration may be necessary in some cases. The SCF solid-phase 

extraction procedure in Sec. 11.4 can be used for this purpose. 

 11.8.2 Extractable organic mercury -- Analyze the eluent from Sec. 11.5.1 by an 

appropriate detection technique. Alternatively, sum the individual organic species 

determined in Sec. 11.6. 

 11.8.3 Extractable inorganic mercury -- Analyze the SCF disk from Sec. 11.5.2 

directly by Method 7473. Alternatively, analyze the eluent from Sec. 11.5.3 by an 

appropriate analytical detection technique. As another alternative, analyze either of the 

sample aliquots from Sec. 11.2.1.4, 11.2.2.7 or 11.5.3 by quantitating the inorganic peak 

as described in Sec. 11.6. 

 11.8.4 Non-extractable mercury -- Analyze the remaining solids from Sec. 

11.2.1.5 or 11.2.2.8 directly by Method 7473 or by suitable decomposition such as Method 

3052 and appropriate analysis procedures. Alternatively, determine the non-extractable 

mercury as the difference between the total mercury concentration (Sec. 11.8.5) and the 

extractable mercury concentration (Sec. 11.8.1). 

 11.8.5 Total mercury -- Analyze the unaltered sample by Method 7473 or by 

suitable decomposition such as Method 3052 and appropriate analysis procedures. 

Alternatively, sum the results from Secs. 11.8.1 and 11.8.4. 

 11.8.6 Semi-mobile mercury -- Analyze the extracted mercury fraction or a 

portion thereof from Sec. 11.7.1.10 using an appropriate detection technique. 

 11.8.7 Non-mobile mercury -- Analyze the remaining solids from Sec. 11.7.1.11 

directly by Method 7473 or by other suitable decomposition such as Method 3052 and 

appropriate analysis procedures. Alternatively, analyze the extracted mercury fraction or a 

portion thereof from Sec. 11.7.2.7 using an appropriate detection technique (Ref. 12, 22). 

12.0 DATA ANALYSIS AND CALCULATIONS 

 12.1 If appropriate for the specific project, calculate the sample dry weight fraction as 

follows: 

1 3

2 3

W W

W W Dry Wt. Fraction

−

−

=

where: 

W1 = Weight of sample + vessel before drying, in g 

W2 = Weight of sample + vessel after drying, in g 
SW-846 Update V 3200 - 19 Revision 1 

 October 2012 

W3 = Weight of empty, dry vessel, in g 

 12.2 The results of the analyses of the various mercury fractions can be converted to 

the concentration of the fraction in the solid sample as follows: 

W S

C V D

Sample Concentration

×

× ×

=

where: 

C = Concentration in the extract in mg/L 

V = Volume of the extract in mL 

D = Dilution factor from the analysis of the extract, if any 

W = Wet weight of original sample aliquot that was extracted, in g 

S = Dry-weight fraction of the sample, g/g (if dry-weight reporting is required) 

 The factors of 1000 in the numerator and the denominator convert mL to L and g to kg, 

effectively canceling each other out. 

13.0 METHOD PERFORMANCE 

 13.1 Performance data and related information are provided in SW-846 methods only as 

examples and guidance. The data do not represent required performance criteria for users of 

the methods. Instead, performance criteria should be developed on a project-specific basis, 

and the laboratory should establish in-house QC performance criteria for the application of this 

method. These performance data are not intended to be and must not be used as absolute QC 

acceptance criteria for purposes of laboratory accreditation.

 13.2 The extractability of pure mercury species are summarized in Table 1 (Ref. 6). 

These data are provided for guidance purposes only.

 13.3 The 2.0% HCl + 10% ethanol extraction procedure was evaluated by spiking 

natural matrices (mercury species spiked at 25 μg mercury per 1 g of sample). The natural 

matrices selected for spiking were silica (SiO2), NIST SRM 2709 (San Joaquin Soil), and two 

soil matrices. The samples were analyzed by Method 7473 and HPLC-ICP-MS. The results are 

summarized in Tables 2 to 5. The "extractable" mercury fraction in 2.0% HCl + 10% ethanol 

extraction is compared with Method 1311 (TCLP) by using BCR 580 and SRM 2709. The 

results are summarized in Table 6. These data are provided for guidance purposes only. 

 13.4 The SCF solid-phase extraction was optimized. The optimal amount of SCF 

packed in column was determined to be 0.2 g with a bed volume of 0.7 mL/0.1 g SCF. The best 

pH range was 3 ± 1. The optimal flow rate was 1.0 mL/min. The optimal eluent 1 was a 1.0 M 

HCl + 1.0 M NaCl solution which minimizes the inorganic mercury interference on the organic 

mercury measurement. The optimal eluent 2 was a solution of 6 M HCl, saturated NaCl and 

0.1% CuCl2⋅2H2O. The overall performance of SCF solid-phase extraction procedure is 

summarized in Table 7. See Ref. 6 for details. These data are provided for guidance purposes 

only. 

 13.5 This method was validated with Institute for Reference Materials and 

Measurements (IRMM) reference material BCR 580. The results are summarized in Table 8. 

The samples were analyzed by EPA Method 7473 and HPLC-ICP-MS (Ref. 6). These data are 

provided for guidance purposes only. 
SW-846 Update V 3200 - 20 Revision 1 

 October 2012 

 13.6 The microwave-assisted extraction method was validated with two specifically 

prepared reference soil materials, three National Institute of Standards and Technology (NIST) 

standard reference materials (SRM 1941a, SRM 2704 and SRM 2709) and one IRMM reference 

material (BCR 580). The samples were analyzed by EPA Method 7473 and HPLC-ICP-MS. 

The results are summarized in Table 9 (Ref. 16, 17). These data are provided for guidance 

purposes only. 

 13.7 The microwave-assisted extraction method was also validated by using EPA 

Method 6800. These results indicate that, based on the matrices tested, the Method 3200 

protocol does not induce any transformation from methylmercury to inorganic mercury or vice 

versa. The results are summarized in Table 10. See Ref. 16, 17. These data are provided for 

guidance purposes only. 

 13.8 The microwave-assisted extraction method was validated by mass balance, that is 

to say, all species were added up to equal the total mercury content in the samples by analysis 

of two crude oil samples using EPA Method 6800. The extractable and non-extractable 

fractions of mercury species were summed to calculate the total mercury content in those 

samples. Method 3200 was used to extract the extractable mercury species which consisted of 

inorganic, methyl-mercury and ethyl-mercury. The total mercury contents were determined by 

Method 6800 SIDMS after digestion with Method 3052. These results indicate that Method 

3200 is suitable for mass balance study of mercury species. Non-extractable mercury was 

spiked in the extracted sample by Method 6800 IDMS and thermally converted to metallic 

mercury, then subsequently analyzed (Ref. 26). In addition, this research paper demonstrates 

that Method 3200, in combination with method 6800, facilitates measurement of mercury 

species’ concentrations and including corrections of the interconversions among these mercury 

species and achieves mass balance of the mercury species. Mass balance is the independent 

measurement of total mercury and its comparison, by adding up all mercury species, a value 

that is compared and confirmed by the independent total mercury measurement. Table 11 and 

Reference 26 demonstrate that the mercury species in the sample equal the total, within the 

stated confidence interval, to the summed measurements; that is the 95% confidence interval in 

the case of the referenced research paper. The results are summarized in Table 11. These 

data are provided for guidance purposes only. 

 13.9 Both extraction protocols outlined in Method 3200 were demonstrated and 

validated by analyzing human hair reference material (IAEA-085). The results are summarized 

in Table 12 and Ref. 27. These results demonstrate that both extraction protocols are capable 

of extracting approximately 100% of several extractable mercury species from human hair, such 

as inorganic and methylmercury and that when Method 3200 is coupled with Method 6800 to 

correct for these species conversions, mass balance is achievable. These data are provided for 

guidance purposes only. 

 13.10 The microwave-assisted extraction method was demonstrated and validated by 

analyzing tuna fish reference material (ERM-CE-464). The tuna fish SRM was extracted and 

analyzed by using HPLC-ICP-MS. The percentage recovery of both mercury species was 

approximately 80% of the certified value. However, when the SRM was spiked with isotopically 

enriched analogues of mercury species and Method 6800 SIDMS is used for quantification, both 

mercury species were corrected to certified values. These data and those in related 

publications demonstrate that after equilibration (as described in Method 6800), the level of 

analyte recovery is no longer a factor affecting accuracy when Method 3200 is used in 

combination with Method 6800. The results are summarized in Table 13 (See Ref. 28). These 

data are provided for guidance purposes only. 
SW-846 Update V 3200 - 21 Revision 1 

 October 2012 

14.0 POLLUTION PREVENTION 

 14.1 Pollution prevention encompasses any technique that reduces or eliminates the 

quantity and/or toxicity of waste at the point of generation. Numerous opportunities for pollution 

prevention exist in laboratory operations. The EPA has established a preferred hierarchy of 

environmental management techniques that places pollution prevention as the management 

option of first choice. Whenever feasible, laboratory personnel should use pollution prevention 

techniques to address their waste generation. When wastes cannot be feasibly reduced at the 

source, the Agency recommends recycling as the next best option. 

14.2 For information about pollution prevention that may be applicable to laboratories 

and research institutions consult Less is Better: Laboratory Chemical Management for Waste 

Reduction, a free publication available from the American Chemical Society (ACS), Committee 

on Chemical Safety, 

http://portal.acs.org/portal/fileFetch/C/WPCP_012290/pdf/WPCP_012290.pdf. 

15.0 WASTE MANAGEMENT 

 15.1 The Environmental Protection Agency requires that laboratory waste management 

practices be conducted consistent with all applicable rules and regulations. The Agency urges 

laboratories to protect the air, water, and land by minimizing and controlling all releases from 

hoods and bench operations, complying with the letter and spirit of any sewer discharge permits 

and regulations, and by complying with all solid and hazardous waste regulations, particularly 

the hazardous waste identification rules and land disposal restrictions. For further information 

on waste management, consult the ACS publication listed in Sec. 14.2. 

 15.2 All inorganic mercury and organomercury waste should be stored separately in 

dedicated glass bottles. The waste bottles should be kept in a hood or ventilated area in 

containers equipped with absorbent pads. Gloves, pipette tips, etc., which might have come in 

contact with mercury containing compounds should be disposed in a dedicated waste 

receptacle fitted with a tight lid. 

REFERENCES 

1. Bloom, N. S.; Gill, G. A.; Cappellino, S.; Dobbs, C.; McShea, L.; Driscoll, C.; Mason, R.; 

Rudd, J., "Speciation and Cycling of Mercury in Lavaca Bay, Texas, Sediments." Environ. 

Sci. Technol., 1999, 33, (1), 7-13. 

2. Templeton, D.; Ariese, F.; Cornelis, R.; Danielsson, L. G.; Muntau, H.; van Leeuvan, H. P.; 

Lobinski, R., "Guidelines for terms related to chemical speciation and fractionation of 

elements: Definitions, structural aspects, and methodological approaches." Pure Appl. 

Chem., 2000, 72, (8), 1453-1470. 

3. Hintelmann, H.; Falter, R.; Ilgen, G.; Evans R. D., "Determination of Artifactual Formation 

of Monomethylmercury (CH3Hg+

) in Environmental Samples Using Stable Hg2+ Isotopes 

with ICP-MS Detection: Calculation of Contents Applying Species Specific Isotope 

Additions." Fresenius J. Anal. Chem., 1997, 358, (3), 363-370. 

4. Bloom, N. S.; Coleman, J. A.; Barber, L., "Artifact formation of methyl mercury during 

aqueous distillation and alternative techniques for the extraction of methyl mercury from 

environmental samples." Fresenius J. Anal. Chem., 1997, 358, (3), 371-377. 

5. Rahman, G. M. M.; Kingston, H. M. S.; Bhandari, S., "Synthesis and Characterization of 
SW-846 Update V 3200 - 22 Revision 1 

 October 2012 

Isotopically Enriched Methylmercury (CH3

201Hg+

)." Appl. Organomet. Chem., 2003, 17, 

(12), 913-920. 

6. Han, Y.; Kingston, H. M.; Boylan, H. M.; Rahman, G. M. M.; Shah, S.; Richter, R. C.; Link, 

D. D.; Bhandari, S., "Speciation of mercury in soil and sediment by selective solvent and 

acid extraction." Anal. Bioanal. Chem., 2003, 375, (3), 428-436. 

7. Huo, D.; Kingston, H. M. S.; Larget, B., "Application of Isotope Dilution in Elemental 

Speciation: Speciated Isotope Dilution Mass Spectrometry (SIDMS)." In: Comprehensive 

Analytical Chemistry, Volume 33 (Elemental Speciation: New Approaches for Trace 

Element Analysis), Elsevier, New York, 2000; 277-313. 

8. Rahman, G. M. M.; Kingston, H. M. S., "Application of Speciated Isotope Dilution Mass 

Spectrometry to Evaluate Extraction Methods for Determining Mercury Speciation in Soils 

and Sediments." Anal. Chem., 2004, 76, (13), 3548-3555. 

9. Rahman, G. M. M.; Kingston, H. M. S.; Kern, J. C.; Hartwell, S. W.; Anderson, R. F.; Yang, 

S. -Y., "Inter-laboratory Validation of EPA Method 3200 for Mercury Speciation Analysis 

using Prepared Soil Reference Materials." Appl. Organomet. Chem., 2005, 19, 301-307. 

10. Labor, U.S. Department of, Occupational Health Guideline for Organo (Alkyl) Mercury, 

Occupational Safety and Health Adminstration, Washington, D.C., 1978. 

11. Nierenberg, D. W.; Nordgren, R. E.; Chang, M. B.; Siegler, R. W.; Blayney, M. B.; 

Hochberg, F.; Toribara, T. Y.; Cernichiari, E.; Clarkson, T., "Delayed Cerebellar Disease 

and Death after Accidental Exposure to Dimethylmercury." New Engl. J. Med., 1993, 338, 

1672-1675. 

12. Kingston, H. M.; Haswell, S. J., Microwave Enhanced Chemistry: Fundamentals, Sample 

Preparation, and Applications, 10th ed.; American Chemical Society, Washington, DC, 

1997. 

13. Applied Isotope Technologies, Inc., www.sidms.com, e-mail: info@sidms.com. 

14. Kingston, H. M. Speciated Isotope Dilution Mass Spectrometry of Reactive Species and 

Related Methods. US Patent 6 790 673 B1, September 14, 2004. 

15. Jain, W.; McLeod, C. W., "Rapid Sequential Determination of Inorganic Mercury and 

Methylmercury in Natural Waters by Flow-injection - Cold Vapor-Atomic-Fluorescence 

Spectrometry." Talanta, 1992, 39, (11), 1537-1542. 

16. Rahman, G. M. M.; Kingston, H. M. S., "Development of Microwave-Assisted Extraction 

Method and Isotopic Validation of Mercury Species in Soils and Sediments." J. Anal. At. 

Spectrom., 2005, 20, 183-191. 

17. Rahman, G. M. M. "Development, Research and Validation of Environmental Speciation 

Methods: Evaluation by Speciated Isotope Dilution Mass Spectrometry in Mercury and 

Chromium Speciation Analysis." Ph. D. Dissertation, Duquesne University, Pittsburgh, PA, 

2004. 

18. Fischer, E.; Tu, Q.; Nagourney, S.; England, R.; Buckley, B. "Microwave-assisted Solvent 

Extraction for Quantitative Simultaneous Extraction of Inorganic Mercury and 

Methylmercury from Soils." 20th Annual National Environmental Monitoring Conference, 

Washington, DC, July 19-23, 2004; Washington, DC, July 19-23, 2004. 
SW-846 Update V 3200 - 23 Revision 1 

 October 2012 

19. Jain, W.; McLeod, C. W., "Field Sampling Technique for Mercury Speciation." Anal. Proc.,

1991, 293-294. 

20. Lee, Y. H.; Mowrer, J., "Determination of Methylmercury in Natural Waters at the Subnanograms-per-litre Level by Capillary Gas Chromatography after Adsorbent Preconcentration." Anal. Chim. Acta, 1989, 221, (2), 259-268. 

21. Mena, M. L.; McLeod, C. W., "Mercury Species Immobilized on Sulphydryl Cotton: A New 

Candidate Reference Material for Mercury Speciation." Mikrochim. Acta., 1996, 123, (1-4), 

103-108. 

22. Boylan, H. M.; Richter, R. C.; Kingston, H. M.; Ricotta, A. C., "Rapid Analysis for the Field: 

Method Development and Application to Natural Gas Utility Sites." Water Air Soil Poll.,

2001, 127, (1-4), 255-270. 

23. Davis, A. Bloom, N. S.; Hee, Q.; Shane, S., "The Environmental Geochemistry and 

Bioaccessibility of Mercury in Soils and Sediments-A Review." Risk Anal., 1997, 17, (5), 

105-113. 

24. Revis, N. W.; Osborne, T. R.; Holdworth, G.; Hadden, C., "Distribution of Mercury Species 

in Soil from a Mercury-Contaminated Site." Water Air Soil Poll., 1989, 17, (5), 557-569. 

25. Revis, N. W.; Osborne, T. R.; Sedgely, D.; King, A., "Quantitative Method for Determining 

the Concentration of Mercury (II) Sulphide in Soils and Sediments." Analyst, 1989, 114, 

823-826. 

26. Rahman, G. M.; Fahrenholz, T. M.; Kingston, H. M. Skip; Pamuku, M.; Hwang, J. D.; 

Young, L.A., "Speciation of Mercury in Crude Oil uisng Speciated Isotope Dilution Mass 

Spectrometry." Spectroscopy, 2010, 25(1), 36-45. 

27. Rahman, G.M.; Fahrenholz, T.M.; Kingston, H. M. Skip, "Application of Speciated Isotope 

Dilution Mass Spectrometry to Evaluate Methods for Efficiencies, Recoveries, and 

Quantification of Mercury Species Transformation in Human Hair." J. Anal. At. Spectrom.,

2009, 24, 83-92. 

28. Reyes, L. H.; Rahman, G. M.; Fahrenholz, T.; Kingston, H. M. Skip, "Comparison of 

Methods with Respect to Efficiencies, Recoveries, and Quantitation of Mercury Species 

Interconversions in Food Demonstrated using Tuna Fish." Anal. Bioanal. Chem., 20008, 

390, 2123-2132. 

17.0 TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA 

 The following pages contain the tables and figures referenced by this method. 
SW-846 Update V 3200 - 24 Revision 1 

 October 2012 

TABLE 1 

EXTRACTABILITY OF MERCURY SPECIES 

 Extractability (%)

Species* Operationallydefined 

Mercury 

Fraction

2.0% HCl + 

10% Ethanol; 

60 °C and 

sonication 

for 30 min

1:2 HNO3: 

reagent water;

95 °C for 40 

min 

1:6:7 HCl:HNO3: reagent 

water; 

95 °C for 40 min

CH3HgCl Extractable 

Organic 

98 99 104

C2H5HgCl 96 94 93

HgCl2 Extractable 

Inorganic 

96 99 99

HgO 97 97 102 

Hg Semi-mobile 4 95 102

HgS Non-mobile 0.15 0.04 97

Hg2Cl2 1.6 11 96

*10 mg of each pure mercury species in each 10-mL extract solutions 

Data obtained from Ref. 6. 

These data are provided for guidance purposes only.

TABLE 2 

RECOVERIES OF EXTRACTABLE MERCURY SPECIES SPIKED INTO A SILICA MATRIX 

Mercury spiked species Recovery range* (%) HPLC-ICP-MS 

HgCl2 96 ± 2

CH3HgCl 98 ± 6

C2H5HgCl 95 ± 7

HgO 98 ± 4

*95% confidence level, n = 3. 

Data obtained from Ref. 6. 

These data are provided for guidance purposes only.
SW-846 Update V 3200 - 25 Revision 1 

 October 2012 

TABLE 3 

RECOVERIES OF EXTRACTABLE MERCURY SPECIES 

SPIKED INTO SRM 2709 SOIL MATRIX 

Mercury spiked species Recovery* (%) HPLC-ICP-MS Recovery* (%) Method 7473

HgCl2 98 ± 7 Total extractable, 92 ± 9

CH3HgCl 92 ± 17

C2H5HgCl 92 ± 17

*95% confidence level, n=3 

Data obtained from Ref. 6. 

These data are provided for guidance purposes only.

TABLE 4 

RECOVERIES OF EXTRACTABLE MERCURY SPECIES SPIKED 

INTO NATURAL SOIL MATRIX 1 

Mercury spiked species Recovery* (%) HPLC-ICPMS

Recovery* (%) Method 7473

HgCl2 106 ± 15 92.5 ± 0.5

CH3HgCl 104 ± 10 95.5 ± 2.5

C2H5HgCl 92 ± 18 91.0 ± 3.0

HgO NA 93.0 ± 1.0

*95% confidence interval with n = 3 

The soil matrix 1 was a soil collected from waste deposit field and sieved through 60- 

mesh screen, then heated in a 240 °C oven for one week. The mercury residue in the soil 

matrix was less than 30 ng mercury per g of soil. 

Data obtained from Ref. 6. 

These data are provided for guidance purposes only.

TABLE 5 

RECOVERIES OF EXTRACTABLE MERCURY SPECIES SPIKED 

INTO NATURAL SOIL MATRIX 2 

Mercury spiked species Recovery* (%) HPLC-ICP-MS Recovery* (%) Method 

7473

HgCl2 12 ± 6 Total extractable, 62 ± 8

CH3HgCl 89 ± 12

C2H5HgCl 95 ± 15

*95% confidence interval, n=3 

The low recovery of the HgCl2 is probably due to complexing of the Hg2+ with components of the 

sample matrix.Data obtained from Ref. 6. 

These data are provided for guidance purposes only.
SW-846 Update V 3200 - 26 Revision 1 

 October 2012 

TABLE 6 

COMPARISON OF 2.0%HCl+10% ETHANOL EXTRACTION WITH METHOD 1311 (TCLP) 

FOR EXTRACTABLE MERCURY SPECIES 

Reference Material 2.0% HCl + 10% Ethanol extractiona Method 1311b

BCR 580 1.4 ± 0.4 ppm 3 ± 2 ppb

SRM 2709 40 ± 8 ppb 12 ± 9 ppb

a95% confidence interval, n=3. 

bMethod 1311 was scaled down to a 1-gram sample size

 Data obtained from Ref. 6. These data are provided for guidance purposes only. 

TABLE 7 

RECOVERIES OF EXTRACTABLE MERCURY SPECIES 

AFTER SCF SOLID-PHASE EXTRACTION 

Eluent CH3HgCl C2H5HgCl HgCl2

Pass-through (10% Ethanol) <0.5% <0.5 <0.5%

1.0 M HCl + 1.0 M NaCl 96% 99% <0.1%

6 M HCl + saturated NaCl + 0.1% 

CuCl2⋅2H2O

<3% <3% 98%

Residue (in SCF) <DL <DL <DL

DL = detection limit (0.01 ng) 

Data obtained from Ref. 6. 

These data are provided for guidance purposes only.

TABLE 8 

VALIDATION OF THE METHOD USING BCR 580 

Method or 

(Certified Value) 

Methyl 

Mercury as 

Mercury (ppb)

Extractable 

Inorganic Mercury 

(ppm)

Nonextractable 

Mercury (ppm)

Total Mercury 

(ppm)

HPLC-ICP-MS 73.6 ± 6.3a1.4 ± 0.4b133 ± 9.6b134.5 ± 9.6c

SCF-Method 7473 78 ± 27b0.9 ± 0.3b127 ± 7b128 ± 7c

(Certified Value) 70.2 ± 3.4 NA NA 132 ± 3

a95% confidence interval with n = 6 

b95% confidence interval with n = 3 

c95% confidence interval with n = 3 

NA = not analyzed 

Data obtained from Ref. 6. 

These data are provided for guidance purposes only.
SW-846 Update V 3200 - 27 Revision 1 

 October 2012 

TABLE 9 

VALIDATION OF MICROWAVE-ASSISTED EXTRACTION METHOD 

Sample Certified / ‘Made-to’ 

Value (μg/g) 

Method 

7473 

(direct 

analysis) 

(μg/g) 

HPLC-ICP-MS 

(μg/g) 

Hg2+ CH3Hg+ Total Hg2+ CH3Hg+ Total 

Material-1 

Inorganic 

Mercury

4.0 ---- 4.08 ±

0.16 

4.26 ± 0.17 ND 4.26 ± 0.17 

Organic 

Mercury

---- 4.0 3.58 ±

0.27 

ND 3.81 ± 0.20 3.81 ± 0.20 

Mixed Mercury 3.0 3.0 5.73 ±

0.58 

3.02 ± 0.06 2.66 ± 0.07 5.68 ± 0.09 

Material-2 

Inorganic 

Mercury

6.0 ---- 6.73 ±

1.04 

6.06 ± 0.56 ND 6.06 ± 0.56 

Organic 

Mercury

---- 6.0 5.44 ±

0.62 

ND 5.94 ± 0.52 5.94 ± 0.52 

Standard Reference Materials 

SRM 1941a 0.5 ± 0.2 ---- 0.61 ±

0.02 

0.67 ± 0.06 ND 0.67 ± 0.06 

SRM 2704 1.44 ±

0.07 

---- 1.51 ±

0.05 

1.40 ± .08 ND 1.40 ± 0.08 

SRM 2709 1.40 ±

0.08 

---- 1.46 ±

0.03 

1.28 ± 0.12 ND 1.28 ± 0.12 

ND = not detectable (ND = 0.05 ng) 

Uncertainties are expressed at the 95% confidence level with n = 3. 

Material-1: 100% processed topsoil; and Material-2: a mixture of 75% processed topsoil and 

25% Ottawa sand 

Data obtained from Ref. 16, 17. 

These data are provided for guidance purposes only.
SW-846 Update V 3200 - 28 Revision 1 

 October 2012 

TABLE 10 

THE DECONVOLUTED CONCENTRATION AND TRANSFORMATION OF MERCURY 

SPECIES IN MATERIAL-1 USING SIDMS CALCULATIONS. 

 Deconvoluted Concentration Interconversion 

 Hg2+ 

(μg/g) 

CH3Hg+ 

(μg/g) 

Hg2+ to CH3Hg+

(%) 

CH3Hg+ to Hg2+

(%) 

DSBE 3.05 ± 0.12 2.69 ± 0.10 1.3 ± 1.5 0.1 ± 1.4 

DSAE 2.94 ± 0.07 2.62 ± 0.09 0.8 ± 1.5 0.7 ± 0.6 

DSBE = double spiked before extraction. DSAE = double spiked after extraction. 

Uncertainties are expressed at the 95% confidence level with n = 3 

Data obtained from Ref. 16, 17. 

These data are provided for guidance purposes only.
SW-846 Update V 3200 - 29 Revision 1 

 October 2012 

TABLE 11 

SIDMS ANALYSIS (6800) OF THREE EXTRACTABLE MERCURY SPECIES AND NONEXTRACTABLE MERCURY FROM CRUDE OIL AFTER EPA METHOD 3200 EXTRACTION 

Sample 

Name 

Extractable Mercury Species, 

μg/kg 

Non-Extractable Mercury 

Species, μg/kg 

Total Mercury*, 

μg/kg 

 Hg2+ MeHg+ EtHg+ 

Serial # 

871833 

17.3 ± 

3.6 

0.8 ± 

0.1 

10.7 ± 

1.1 

342.5 ± 46.8 

370.2 ± 47.0 (414.1 

± 24.7) 

Serial # 

870987 

23.6 ± 

3.7 

0.5 ± 

0.1 

7.4 ± 

2.2 

323.3 ± 30.7 

354.8 ± 31.0 

(437.8 ± 25.4) 

Uncertainties are at 95% CL, n = 12 (3 x 4). 

*values in parentheses are total mercury determined by isotope dilution mass spectrometry 

(IDMS) of the crude oil after Method 3052 digestion. 

Data obtained from Ref. 26. 

These data are provided for guidance purposes only.

TABLE 12 

MERCURY SPECIATION IN HUMAN HAIR CERTIFIED REFERENCE MATERIAL, IAEA-085, BY 

HPLC-ICP-MS AND QUANTIFICATION BY EXTERNAL CALIBRATION.a THE VALUES ARE 

MEANS ± 95% CL (n = 4 x 4)b

 

Extraction procedure Hg2+ (as Hg) 

(μg/g) 

CH3Hg+ (as Hg) 

(μg/g) 

Sum of Species 

(μg/g) 

Mean degree of 

Transformation (%) 

Hg2+ to 

CH3Hg+

CH3Hg+

 to 

Hg2+

Method 3200 

(ultrasonication) 0.33 ± 0.06 20.87 ± 0.37 

(91 ± 2) 

21.21 ± 0.38 

(91 ± 2) ----- ----- 

Method 3200 

(ultrasonication) and 

Method 6800 

1.13 ± 0.25 19.80 ± 1.25 

(87 ± 6) 

20.93 ± 1.28 

(90 ± 6) 9 ± 3 0 ± 1 

Method 3200 (MAE) 1.80 ± 0.61 22.67 ± 1.54 

(99 ± 7) 

24.67 ± 1.65 

(106 ± 7) ----- ----- 

Method 3200 (MAE) 

and Method 6800 0.59 ± 0.22 23.65 ± 1.42 

(103 ± 6) 

24.24 ± 1.44 

(105 ± 6) 4 ± 2 6 ± 1 

aCertified total Hg is = 23.2 ± 0.8 μg/g, certified CH3Hg+

 is = 22.9 ± 1.0 μg/g and estimated Hg2+ is 0.1 – 

0.5 μg/g. bThe percent recoveries of total Hg and CH3Hg+

 are indicated in parentheses. 

Data obtained from Ref. 27. 

These data are provided for guidance purposes only.
SW-846 Update V 3200 - 30 Revision 1 

 October 2012 

TABLE 13 

MERCURY SPECIATION IN TUNA FISH CERTIFIED REFERENCE MATERIAL (ERM-CE-464) 

BY METHOD 6800 HPLC-ICP-MS AFTER EXTRACTION WITH METHOD 3200 WITH AND 

WITHOUT SPIKING ISOTOPICALLY ENRICHED ANALOGUES. 

 Hg2+ (mg/kg) CH3Hg+

 (mg/kg) Sum of Species 

(mg/kg) 

Degree of transformation, 

mean 

Hg2+ to 

CH3Hg+

CH3Hg+

 to 

Hg2+

Method 3200 0.06 ± 0.04 3.94 ± 0.12 

(77 ± 2) 

4.00 ± 0.13 

(76 ± 2) ------ ------ 

Method 3200 

and Method 

6800 

0.11 ± 0.07 5.60 ± 0.33 

(109 ± 6) 

5.71 ± 0.34 

(109 ± 6) 18 ± 4 0.8 ± 0.6 

Certified total Hg is 5.24 ± 0.10 mg/kg, certified CH3Hg+ is 5.12 ± 0.16 mg/kg and estimated Hg2+ is 0.12 

mg/kg. The percent recoveries of total Hg and CH3Hg+

 are indicated in parentheses. 

Isotopic spiking as described in Method 6800 prior to extraction with Method 3200 

Data obtained from Ref.28. 

These data are provided for guidance purposes only.
SW-846 Update V 3200 - 31 Revision 1 

 October 2012 

FIGURE 1 

METHYL MERCURY TRANSFORMATION 

Solid line: A methyl mercury standard in acid solution before the addition of iron 

Dash line: A methyl mercury standard in acid solution with 500 ppm iron. 

NOTE: Transformation observed 8 hr after spiking (HPLC flow rate 0.8 mL/min). 

Figure taken from Ref. 6. 

These data are provided for guidance purposes only.
SW-846 Update V 3200 - 32 Revision 1 

 October 2012 

FIGURE 2 

Solid line: An ethyl mercury standard in acid solution without the addition of iron 

Dash line: An ethyl mercury standard in acid solution with 500 ppm iron. 

NOTE: Transformation observed 8 hr after spiking (HPLC flow rate 0.8 mL/min). 

Figure taken from Ref. 6. 

These data are provided for guidance purposes only.
SW-846 Update V 3200 - 33 Revision 1 

 October 2012 

METHOD 3200 

MERCURY SPECIES FRACTIONATION AND QUANTIFICATION BY MICROWAVE 

ASSISTED EXTRACTION, SELECTIVE SOLVENT EXTRACTION AND/OR SOLID PHASE 

EXTRACTION 

11.8.7 Analysis

11.8.3 Analysis

11.8.7 Analysis

11.8.3 Analysis

11.8.2 Analysis

11.8.6 Analysis

11.8.1 Analysis

11.8.4 Analysis 11.8.1 Analysis

11.2.1.5 or 11.2.2.8

11.7.1.10

Semi-mobile

Hg extract

11.7.1.11

Non-mobile

Hg residue

11.7.1 Extraction

11.7.2.7

Non-mobile

Hg extract

Total Hg

11.4 SCF 

Solid-phase

extraction

11.4.6

Extractable Hg

on SCF disk

11.5.1

Extractable

Organic Hg

concentrate

11.5.2

Extractable

Inorganic Hg

on SCF disk

11.5.1 Elution

of SCF

11.5.3 Elution

of SCF

11.5.3

Extractable

Inorganic Hg

concentrate

11.2.1 Microwave

extraction

11.2.2 Ultrasound

extraction

11.7.2

Extraction

11.2.1.4 or 11.2.2.7

Extractable 

Hg extract

Non-extractable 

Hg residue

11.8.7 Analysis

11.8.3 Analysis

11.8.7 Analysis

11.8.3 Analysis

11.8.2 Analysis

11.8.6 Analysis

11.8.1 Analysis

11.8.4 Analysis 11.8.1 Analysis

11.2.1.5 or 11.2.2.8

11.7.1.10

Semi-mobile

Hg extract

11.7.1.11

Non-mobile

Hg residue

11.7.1 Extraction

11.7.2.7

Non-mobile

Hg extract

Total Hg

11.4 SCF 

Solid-phase

extraction

11.4.6

Extractable Hg

on SCF disk

11.5.1

Extractable

Organic Hg

concentrate

11.5.2

Extractable

Inorganic Hg

on SCF disk

11.5.1 Elution

of SCF

11.5.3 Elution

of SCF

11.5.3

Extractable

Inorganic Hg

concentrate

11.2.1 Microwave

extraction

11.2.2 Ultrasound

extraction

11.7.2

Extraction

11.2.1.4 or 11.2.2.7

Extractable 

Hg extract

Non-extractable 

Hg residue
SW-846 Update V 3200 - 34 Revision 1 

 October 2012 

Appendix A: 

Summary of Revisions to Method 3200 (as compared to previous Revision 0, July 2005)

1. Improved overall method formatting for consistency with new SW-846 methods style 

guidance. 

2. Updated and expanded “QUALITY CONTROL” section for better adherence to current 

SW-846 method guidelines and for improved alignment with current universal practices 

for published analytical methods. 

3. Additional comment added in Sec. 8.3 concerning holding times. 

4. Minor editorial and technical revisions were made throughout to improve method clarity.