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METHOD 245.6
DETERMINATION OF MERCURY IN TISSUES
BY COLO VAPOR ATOMIC ABSORPTION SPECTROMETRY
Edited by Larry B. Lobring and Billy B. Potter
Inorganic Chemistry Branch
Chemistry Research Division
Revision 2.3
April 1991
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ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
281 HEADQUARTERS LIBRARY
ENVIRONMENTAL PROTECTIOM
WASHINGTON, D.C ^f-"
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METHOD 245.6
DETERMINATION OF MERCURY IN TISSUES
BY COLD VAPOR ATOMIC ABSORPTION SPECTROMETRY
1- SCOPE AND APPLICATION
1.1 This procedure measures total mercury (organic + inorganic) in
biological tissue samples.
1.2 The range of the method is 0.2 to 5 M9/9- The range may be extend-
ed above or below the normal range by increasing or decreasing
sample size or by optimizing instrument sensitivity.
2. SUMMARY OF METHOD
2,1 A weighed portion of the tissue sample is digested with sulfuric and
nitric acid at 58°C followed by overnight oxidation with potassium
permanganate and potassium persulfate at room temperature. Mercury
in the digested sample is reduced with stannous chloride to
elemental mercury and measured by the conventional cold vapor atomic
absorption technique.
3. DEFINITIONS
3.1 BIOCHEMICAL OXYGEN DEMAND (BOD) BOTTLE - BOD bottle, 300 ± 2 mL with
a ground glass stopper or an equivalent flask, fitted with a ground
.glass stopper.
3.2 CALIBRATION BLANK - A volume of ASTM type II reagent water prepared
in the same manner (acidified) as the calibration standard.
3.3 CALIBRATION STANDARD (CAL) - A solution prepared from the mercury
stock standard solution used to calibrate the instrument response
with respect to analyte concentration.
3.4 INSTRUMENT DETECTION LIMIT (IDL) - The mercury concentration that
produces a signal equal to three times the standard deviation of the
blank signal.
3.5 LABORATORY FORTIFIED BLANK (LFB) - An aliquot of ASTM type II
reagent water to which known quantities of inorganic and/or organic
mercury are added in the laboratory. The LFB is analyzed exactly
like a sample, and its purpose is to determine whether method
performance Is within accepted control limits.
3.6 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) - A portion of a tissue
sample to which known quantities of calibration standard are added
in the laboratory. The LFM is analyzed exactly like a sample, and
its purpose is to determine whether the sample matrix contributes
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bias to the analytical results. The background concentrations of
the analytes in the sample matrix must be determined in a separate
aliquot and the measured values in the LFM corrected for the
concentrations found,
3.7 LABORATORY REAGENT BLANK (LRB) - An aliquot of ASTM type II reagent
water that is treated exactly as a sample including exposure to all
glassware, equipment, and reagents used in analyses. The LRB is
used to determine if method analyte or other interferences are
present in the laboratory environment, the reagents or apparatus.
3.8 LINEAR DYNAMIC RANGE (LDR) - The concentration range over which the
analytical working curve remains linear.
3.9 METHOD DETECTION LIMIT (MDL) - The minimum concentration of mercury
that can be identified, measured and reported with 99% confidence
that the analyte concentration is greater than zero and determined
from analysis of laboratory fortified tissue sample matrix (LFM).
3.10 QUALITY CONTROL SAMPLE (QCS) - A tissue sample containing known
concentration of mercury derived from externally prepared test
materials. The QCS is obtained from a source external to the
laboratory and is used to check laboratory performance.
3.11 TISSUE SAMPLE - A biological sample matrix exposed to a marine,
brackish or fresh water environment. It is limited fay this method
to the edible tissue portion.
3.12 STOCK STANDARD SOLUTION - A concentrated solution containing mercury
prepared in the laboratory using assayed mercuric chloride or stock
standard solution purchased from a reputable commercial source.
4. INTERFERENCES
4.1 Interferences have been reported for waters containing sulfide,
chloride, copper and tellurium. Organic compounds which have broad
band UV absorbance (around 253.7 nm) are confirmed interferences.
The concentration levels for interferants are difficult to define.
This suggests that quality control procedures (Sect. 10) must be
strictly followed.
4.2 Volatile materials which absorb at 253.7 nm will cause a positive
interference. In order to remove any interfering volatile
materials, the dead air space in the BOO bottle should be purged
before the addition of stannous chloride solution.
4.3 Interferences associated with the tissue matrix are corrected for in
calibration procedure (Sect. 9).
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5. SAFETY
5.1 The toxicity and cardnogenicity of each reagent used in this method
has not been fully established. Each chemical should be regarded as
a potential health hazard and exposure to these compounds should be
minimized by good laboratory practices . Normal accepted
laboratory safety practices should be followed during reagent
preparation and instrument operation. Always wear safety glasses or
full-face shield for eye protection when working with these
reagents. Each laboratory is responsible for maintaining a current
safety plan, a current awareness file of OSHA regulations regarding
the safe handling of the chemicals specified in this method ' .
5.2 Mercury compounds are highly toxic if swallowed, inhaled, or
absorbed through the skin. Analyses should be conducted in a
laboratory exhaust hood. The analyst should use chemical resistant
gloves when handling concentrated mercury standards.
5.3 All personnel handling tissue samples should beware of biological
hazards associated with tissue samples. Bivalve mollusk may
concentrate toxins and pathogenic organisms. Tissue dissection
should be conducted in a bio-hazard hood and personnel should wear
surgical mask and gloves.
6. APPARATUS AND EQUIPMENT
6.1 ABSORPTION CELL - Standard spectrophotometer cells 10-cm long,
having quartz windows may be used. Suitable cells may be
constructed from plexiglass tubing, 1-in. O.p. by 4-1/2-in. long.
The ends are ground perpendicular to the longitudinal axis and
quartz windows (1-in- diameter by 1/16-in. thickness) are cemented
in place. Gas inlet and outlet ports (also of plexiglass but 1/4-
in. O.D.) are attached approximately 1/2-in. from each end. The
cell is strapped to a burner for support and aligned in the light
beam to give the maximum transmittance.
6.2 AERATION TUBING - Inert mercury-free tubing is used for passage of
mercury vapor from the sample bottle to the absorption cell. In
some systems, mercury vapor is recycled. Straight glass tubing
terminating in a coarse porous glass aspirator is used for purging
mercury released from the tissue sample in the BOO bottle.
6.3 AIR PUMP - Any pump (pressure or vacuum system) capable of passing
air at 1 L/min is used. Regulated compressed air can be used in an
open one-pass system.
6.4 ATOMIC ABSORPTION SPECTROPHOTOMETER - Any atomic absorption unit
having an open sample presentation area in which to mount the
absorption cell is suitable. Instrument settings recommended by the
particular manufacturer should be followed. Instruments designed
specifically for mercury measurement using the cold vapor technique
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are commercially available and may be substituted for the atomic
absorption spectrophotometer.
6.5 BIOCHEMICAL OXYGEN DEMAND (BOD) BOTTLE - See Sect. 3.1.
6.6 DRYING TUBE - Tube (6-in. x 3/4-in. OD) containing 20 g of magnesium
perchlorate. The filled tube is Inserted (in-line) between the BOD
bottle and the absorption tube. In place of the magnesium
perchlorate drying tube, a small reading lamp is positioned to
radiate heat (about 10°C above ambient) on the absorption cell.
This avoids water condensation in the cell.
6.7 FLOWMETER - Capable of measuring an air flow of 1 L/min.
6.8 MERCURY HOLLOW CATHODE LAMP - Single element hollow cathode lamp or
electrodeless discharge lamp and associated power supply.
6.9 RECORDER - Any raulti-range variable speed recorder that is
compatible with the UV detection system is suitable.
6.10 WATER BATH - The water bath should have a covered top and capacity
to sustain a water depth of 2-1n. to 3-in. at 95*C ± 1°C. The
dimensions of the water bath should be large enough to accommodate
BOD bottles containing CAL, LFB, LFM, LRB, QCS and tissue samples
with the lid on.
REAGENTS AND CONSUMABLE MATERIALS
7.1 Reagents may contain elemental impurities which bias analytical
results. All reagents should be assayed by the chemical
manufacturer for mercury and meet ACS specifications.
7.1.1 Hydroxylamine Hydrochloride (NHjOH-HCl), (CASRN 5470-11-1)
may be used in place of hydroxylamine sulfate in Sect.
7.6. The assayed mercury level of either compound is not
to exceed 0.05 ppm.
7.1.2 Hydroxylamine Sulfate [(NH2OH)2'H2SOJ (CASRN 10039-54-0);
assayed mercury level 1s not to exceed 1 ppb.
7.1.3 Mercuric Chloride (HgCl2), (CASRN 7487-94-7).
7.1.4 Nitric Acid (HN03), concentrated (sp.gr. 1.41), (CASRN
7697-37-2); assayed mercury level is not to exceed 1 ppb.
7.1.5 Potassium Permanganate (KMn04), (CASRN 7722-64-7); assayed
mercury level 1s not to exceed 0.05 ppm.
7.1.6 Potassium Persulfate (K2S2Oa), (CASRN 7727-21-1); assayed
mercury level is not to exceed 0.05 ppm.
7.1.7 Reagent Water, ASTH type II.*
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7.1.8 Sodium Chloride (NaCl), (CASRN 7647-14-5); assayed mercury
level 1s not to exceed 0.05 ppm.
7.1.9 Stannous Chloride (SnCl2'2H20), (CASRN 10025-69-1);
assayed mercury level is not to exceed 0.05 ppra.
7.1.10 Stannous Sulfate (SnSOJ, (CASRN 7488-55-3); assayed
mercury level is not to exceed 0.05 ppm,
7.1.11 Sulfurlc Acid (H2S04), concentrated (sp.gr. 1.84), (CASRN
7664-93-9); assayed mercury level is not to exceed 1 ppb.
7.2 MERCURY CALIBRATION STANDARD - To each volumetric flask used for
serial dilutions, acidify with (0.1 to 0.256 by volume) HNOj
(Sect. 7.1.4). Using mercury stock standard (Sect. 7.3), make
serial dilutions to obtain a concentration of 0.1 /*g Hg/mL. This
standard should be prepared just before analyses.
7.3 MERCURY STOCK STANDARD - Dissolve in a 100-tnL volumetric flask
0.1354 g HgCl, (Sect. 7.1.3) with 75 ml of reagent water
(Sect. 7.1.7). Add 10 ml of cone. HN03 (Sect. 7.1.4) and dilute to
mark. Concentration is 1.0 mg Hg/mL.
7.4 POTASSIUM PERMANGANATE SOLUTION - Dissolve 5 g of KMn04
(Sect. 7.1.5) in 100 ml of reagent water (Sect. 7,1.7).
7.5 POTASSIUM PERSULFATE SOLUTION - Dissolve 5 g of K2S208 (Sect. 7.1.6)
in 100 mL of reagent water (Sect. 7,1.7).
7.6 SODIUM CHLORIDE-HYDROXYLAMINE SULFATE SOLUTION - Dissolve 12 g of
NaCl (Sect. 7.1.8) and 12 g of (NH2OH)2'H2SO, (Sect. 7.1.2) or 12 g
of NHjOH'HCl (Sect. 7.1.1) dilute with reagent water (Sect. 7.1.7)
to 100 mL.
7.7 STANNOUS CHLORIDE SOLUTION - Add 25 g SnCl2'2H20 (Sect. 7.1.9) or
25 g of SnS04 to 250 raL of 0.5 N H2S04 (Sect. 7.8). This mixture is
a suspension and should be stirred continuously during use.
7.8 SULFURIC ACID, 0.5 N - Slowly add 14.0 mL of cone. H2SO.
(Sect. 7.1.10) dilute to 1 L with reagent water (Sect. 7.1.7).
8. SAMPLE COLLECTION. PRESERVATION AND STORAGE
8.1 Because of the extreme sensitivity of the analytical procedure and
the presence of mercury in a laboratory environment, care must be
taken to avoid extraneous contamination. Sampling devices, sample
containers and plastic items should be determined to be free of
mercury; the sample should not be exposed to any condition in the.
laboratory that may result In contact or airborne mercury
contamination.
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8.2 The tissue sample should be preserved and dissected In accordance
with Method 200.3, "Sample Preparation Procedure for Spectrochemical
Determination of Total Recoverable Elements in Biological Tissues",
only Sect. 8. Tissue Dissection, is used in this method.
8.3 Weigh 0.2- to 0.3-g portions of each sample and place in the bottom
of a dry BOD bottle. Care must be taken that none of the sample
adheres to the side of the bottle. Immediately cap and cover the
top of the BOO bottle with aluminum foil.
9« CALIBRATION AND STANDARDIZATION
9.1 The calibration curve is prepared from values determined for
portions of fortified tissue treated in the manner used for the
tissue samples being analyzed. For preparation of the calibration
standards, blend a portion of tissue in a Waring blender.
9.2 Transfer accurately weighed portions to each of five dry BOD
bottles. Each sample should weigh about 0.2 g. Add 4 ml of cone.
H2S04 and 1 ml of cone. HN03 to each bottle and place in a water
bath maintained at 58°C until the tissue is completely dissolved (30
to 60 minutes).
9.3 Cool and transfer 0.5, 2.0, 5.0 and 10.0 mL aliquots of the CAL
(Sect. 7.2) solution containing 0.5 to 1.0 fig of Hg to the BOD
bottles containing tissue. Cool to 4°C in an ice bath and
cautiously add 15 ml of potassium permanganate solution (Sect. 7.4)
and 8 ml of potassium persulfate (Sect. 7.5). Allow to stand
overnight at room temperature under oxidizing conditions.
9.4 Construct a standard curve by plotting peak height or maximum
response of the standard (obtained in Sect, 11.7) versus micrograms
of mercury contained in the bottles. The standard curve should
comply with Sect. 10.2.3. Calibration using computer or calculator
based regression curve fitting techniques on concentration/response
data is acceptable.
10. QUALITY CONTROL
10.1 Each laboratory using this method is required to operate a formal
quality control (QC) program. The minimum requirements of this
program consist of an initial demonstration of laboratory capability
by analyses of laboratory reagent blanks, fortified blanks and
samples used for continuing check on method performance. Standard
Reference Materials (SRMs) • 6 are available and should be used to
validate laboratory performance. Commercially available tissue
reference materials are acceptable for routine laboratory use. The
laboratory is required to maintain performance records that define
the quality of data generated.
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10,2 INITIAL DEMONSTRATION OF PERFORMANCE
10.2.1 The Initial demonstration of performance Is used to
characterize Instrument performance (MOLs and linear
calibration ranges) for analyses conducted by this method.
10.2.2 A mercury HDL should be established using LFH at a
concentration of two to five times the estimated detection
limit7. To determine MDL values, take seven replicate
aliquots of the LFH and process through the entire
analytical method. Perform all calculations defined 1n
the method and report the concentration values in the
appropriate units. Calculate the MDL as follows:
MDL - (t) x (S)
where, t -> Student's t value for a 99X confidence level
and a standard deviation estimate with n-1
degrees of freedom [t = 3.14 for seven
replicates].
S = standard deviation of the replicate analyses.
A MDL should be determined every six months or whenever a
significant change in background or instrument response 1s
expected (e.g., detector change).
10.2.3 Linear calibration ranges - The upper limit of the linear
calibration range should be established for mercury by
determining the signal responses from a minimum of three
different concentration standards, one of which is close
to the upper limit of the linear range. Linear calibration
ranges should be determined every six months or whenever a
significant change in Instrument response is observed.
10.3 ASSESSING LABORATORY PERFORMANCE - REAGENT AND FORTIFIED BLANKS
10.3.1 The laboratory must analyze at least one LRB (Sect. 3.7)
with each set of samples. LRB data are used to assess
contamination from the laboratory environment and to
characterize spectral background from the reagents used in
sample processing. If an mercury value in a LRB exceeds
its determined MDL, then laboratory or reagent
contamination is suspect. Any determined source of
contamination should be corrected and the samples
reanalyzed.
10.3.2 The laboratory must analyze at least one LF6 (Sect. 3.5)
with each batch of samples. Calculate accuracy as percent
recovery (Sect. 10.4.2). If the recovery of mercury falls
outside control limits (Sect. 10.3.3), the method is
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judged out of control. The source of the problem should
be Identified and resolved before continuing analyses.
10.3.3 Until sufficient data (usually a minimum of 20 to 30
analyses) become available, each laboratory should assess
its performance against recovery limits of 85-115%. When
sufficient internal performance data become available,
develop control limits from the percent mean recovery (x)
and the standard deviation (S) of the mean recovery.
These data are used to establish upper and lower control
limits as follows:
UPPER CONTROL LIMIT - x -f 3S
LOWER CONTROL LIMIT - x - 3S
After each five to ten new recovery measurements, new
control limits should be calculated using only the most
recent 20 to 30 data points.
10.4 ASSESSING ANALYTE RECOVERY - LABORATORY FORTIFIED SAMPLE MATRIX
10.4.1 The laboratory must add a known amount of mercury to a
minimum of 10% of samples or one sample per sample set,
whichever is greater. Select a tissue sample that is
representative of the type of tissue being analyzed and
has a low mercury background. It is recommended that this
sample be analyzed prior to fortification. The
fortification should be 20% to 50% higher than the
analyzed value. Over time, samples from all routine
sample sources should be fortified.
10.4.2 Calculate the percent recovery, corrected for background
concentrations measured in the unfortified sample, and
compare these values to the control limits established in
Sect. 10.3.3 for the analyses of LFBs. A recovery
calculation is not required if the concentration of the
analyte added is less than 10% of the sample background
concentration. Percent recovery may be calculated in
units appropriate to the matrix, using the following
equation:
C - C
R = * x 100
where, R = percent recovery
Cs = fortified sample concentration
C = sample background concentration
s » concentration equivalent of
fortifier added to tissue sample.
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10.4.3 If mercury recovery falls outside the designated range,
and the laboratory performance Is shown to be In control
(Sect. 10.3), the recovery problem encountered with the
fortified tissue sample 1s judged to be matrix related,
not system related. The result for mercury 1n the
unfortified sample must be labelled to Inform the data
user that the results are suspect due to matrix effects.
11. PROCEDURE
11.1 Add 4 mL of cone. H2SO, (Sect. 7.1.10} and 1 ml of cone, HNO,
(Sect. 7.1.4) to each Dottle and place In a water bath maintained at
58°C until the tissue is completely dissolved (30 to 60 min).
11.2 Cool to 4°C in an ice bath and cautiously add 5 ml of potassium
permanganate solution (Sect. 7.4) in 1 mL increments. Add an
additional 10 ml or more of permanganate, as necessary to maintain
oxidizing conditions. Add 8 ml of potassium persulfate solution
(Sect. 7.5). Allow to stand overnight at room temperature.
As an alternative to the overnight digestion, tissue solubilization
may be carried out In a water bath at 80°C for 30 min. The sample
is cooled and IB mL of potassium permanganate solution (Sect. 7.4)
added cautiously followed by 8 ml of potassium persulfate solution
(Sect, 7.5). At this point, the sample is returned to the water
bath and digested for an additional 90 min at 30°C. Calibration
standards are treated in the same manner.
11.3 Turn on the spectrophotometer and circulating pump. Adjust the pump
rate to 1 L/min. Allow the spectrophotometer and pump to stabilize.
11.4 Cool the 300 bottles to room temperature and dilute in the following
manner:
11.4.1 To each BOO bottle containing the CAL, LFB and LRB, add 50
mL of reagent water (Sect. 7.1.7).
11.4.2 To each BOD bottle containing a tissue sample, QCS or LFM,
add 55 mL of reagent water (Sect. 7.1.7).
11.5 To each BOO bottle, add 6 mL of sodium chloride-hydroxylamine
sulfate solution (Sect. 7.6) to reduce the excess permanganate.
11.6 Treating each bottle individually:
11.6.1 Placing the aspirator Inside the BOD bottle and above the
liquid, purge the head space (20 to 30 sec) to remove
possible gaseous interferents.
11.6.2 Add 5 mL of stannous chloride solution (Sect. 7.7) and
Immediately attach the bottle to the aeration apparatus.
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11.6.3 The absorbance, as exhibited either on the spectro-
photometer or the recorder, will increase and reach
maximum within 30 sec. As soon as the recorder pen levels
off, approximately 1 min, open the bypass value (or
optionally remove aspirator from the BOD bottle if it Is
vented under the hood) and continue the aeration until the
absorbance returns to its minimum value.
11.7 Close the bypass value, remove the aspirator from the BOD bottle and
continue the aeration. Repeat step (Sect. 11.6) until all BOD
bottles have been aerated and recorded.
12. CALCULATIONS
12.1 Measure the peak height of the unknown from the chart and read the
mercury value from the standard curve.
12.2 Calculate the mercury concentration 1n the sample by the formula:
Wtf Hg/g = \ig fig in the aliquot
wt. of the aliquot in grams
12.3 Report mercury concentrations as follows: Below 0.1 M9/9. <
0.1 /jg/g; between 0.1 and 1 M9/9» to the nearest 0.01 ^g; between 1
and 10 Atg/g, to nearest 0.1 /itg; above 10 /^g/g, to nearest M9-
13. PRECISION AND ACCURACY
13.1 The standard deviation for mercury in fish tissue samples are
reported as 0.19 ± 0.02 fig Hg/g , 0.74 ± 0.05 ng Hg/g and 0.74 ±
0.05 ng Hg/g with recoveries for LFM being 112%, 93%, and 86%,
respectively. These tissue samples were fortified with methyl
mercuric chloride.
14. REFERENCES
1. "Safety in Academic Chemistry Laboratories", American Chemical
Society Publication, Committee on Chemical Safety, 3rd Edition,
1979,
2. "OSHA Safety and Health Standards, General Industry", (29CFR 1910),
Occupational Safety and Health Administration, OSHA 2206, revised
January, 1976,
3. "Proposed OSHA Safety and Health Standards, Laboratories",
Occupational Safety and Health Administration, Federal Register,
July 24, 1986.
4. "Specification for Reagent Water," Annual Book of ASTM Standards,
D1193, Vol. 11.01, 1990.
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5. National Institute of Standards and Technology, Office of Standards
Reference Materials, Gaithersburg, MD Z0899: Aquatic Plant -
Lagarosiphon major (CRM 8030), Aquatic Plant - Platihypnidium
riparioldes (CRM 8031), Oyster Tissue (SRM 1566a), Albacore Tuna
(RM 50).
6. National Research Council of Canada, Marine Analytical Chemistry
Standards Program, Division of Chemistry, Montreal Road, Ottawa,
Ontario K1A OR9, Canada: Dogfish Liver (DOLT-1), Dogfish Muscle
(DORM-1), Non Defatted Lobster Kepatopancreas (LUTS-1), Lobster
Hepatopancreas (TORT-1).
7. Code of Federal Regulations 40, Ch. 1, Pt. 136 Appendix B.
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21012
0 * BUBBLER
ABSORPTION
CELL
SAMPLE SOLUTION
IN BOO BOTTLE
SCRU88ER
CONTAINING
A MERCURY
A8SORBNO
MEDIA
1. Apparatus f«r Fl «*!•$$ torcury
Because of the toxic nature of mercury vapor, Inhalation must be avoided.
Therefore, a bypass has been included in the system to either vent the mercury
vapor into a exhaust hood or pass the vapor through some absorbing media, such
as: a) equal volumes of 0.1 N KHnO, and 10X H2SO,
b) 0.25% iodine In a 3X KI solution.
A specially treated charcoal that will absorb mercury vapor is also available
from Barnebey and Cheney, P.O. Box 2526, Columbus, OH 43216, Catalog No. 580-
13 or 580-22.
293
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