188
shake tube by hand 15 s. Loosen cap and centrifuge 5 min at 2000 rpm. Carefully decant
and discard acetone. (Use dropping pipette to
remove acetone, if necessary.) Repeat 25 mL
acetone wash step 2 more times. Break up tissue with glass stirring rod before shaking
tube, if necessary. Add 20 p.L toluene; tightly
cap and vigorously shake tube by hand 30 s.
Loosen cap and centrifuge 5 min at 2000 rpm.
Carefully decant (or draw off with dropping
pipette) and discard toluene. Extraneous
peaks in final GC chromatogram may indicate that more vigorous shaking with
acetone and toluene is required. In products
for which Me Hg recoveries are to be determined, fortify tissue at this point by adding
working fortification solution, to centrifuge
tubes.
Add 2.5 mL HCI solution, to centrifuge
tube containing acetone and toluene washed
sample. Break up tissue with glass stirring
rod, if necessary. Extract CH3HgCI by adding
20 mL toluene and shaking tube gently but
thoroughly 5 mm on mechanical shaker at
setting 5 (2 min by hand). Loosen cap and
centrifuge 5 min at 2000 rpm. If emulsion is
present after centrifugation, add 1 mL isopropanol and gently stir into toluene layer
with glass stirring rod to reduce emulsion.
Do not mix isopropanol with aqueous phase.
Add equal amounts of isopropanol to blank
and test solutions. If emulsion is not present,
do not add isopropanol to blank or test
solutions. Vigorous mixing of isopropanol
with HCI may produce interfering peaks in
chromatograms. Recentrifuge. With dropping pipette, carefully transfer toluene to
graduated cylinder. Rinse walls of centrifuge
tube with 1-2 mL toluene and transfer rinse
to graduated cylinder. Repeat extraction
step 1 more time. Combine both extracts in
graduated cylinder, dilute to 50 mL with
toluene, stopper and mix well. Add 10 g
Na2S04 and mix again. Tightly stoppered
extracts (sealed with Teflon tape) may be refrigerated and held overnight at this point.
Analyse by GC.
4 The Measurement Process in Chemistry
F. Gas Chromatography
Verify that system is operating properly by
injecting 5 p.L standard solution containing
0.005 p.g HglmL into GC system. Difference
between CH3HgCI peak heights for 2 injections should be ~4%. Check detector
linearity by chromatographing all working
standard solutions.
Inject 5 p.L standard solution with concentration approximately equal to or slightly
greater than concentration of extract. Immediately after CH3HgCI peak appears, inject another 5 p.L extract. Immediately after
CH3HgCI and background peaks for extract
appear, inject another 5 p.L aliquot of standard solution. Because column performance
and peak height slowly decrease with time,
calculate Hg concentration in each test
sample by comparing peak height for each
test extract to average peak height for standard solutions injected immediately after test
extract.
Correct height of CH3HgCI peak for test
extract by subtracting height of peak for
method blank obtained at same attenuation
and recorder sensitivity. Calculate Me-bound
Hg content of test sample expressed as p.g
Hg/g (ppm Hg) by comparing height of peak
from injection of test extract to average
height of peak from duplicate injections of
standard solution as follows:
p.gHg/g fish = (RIR') x (e'le) x 50
where
R = corrected height of CH3HgCI peak from
injection of test extract;
R' = average height of CH3HgCl peak from
duplicate injections of standard solution;
e = weight (g) of test portion;
e' = concentration (p.glmL) of Hg in standard solution; and
50 = fmal volume (mL)
shake tube by hand 15 s. Loosen cap and centrifuge 5 min at 2000 rpm. Carefully decant
and discard acetone. (Use dropping pipette to
remove acetone, if necessary.) Repeat 25 mL
acetone wash step 2 more times. Break up tissue with glass stirring rod before shaking
tube, if necessary. Add 20 p.L toluene; tightly
cap and vigorously shake tube by hand 30 s.
Loosen cap and centrifuge 5 min at 2000 rpm.
Carefully decant (or draw off with dropping
pipette) and discard toluene. Extraneous
peaks in final GC chromatogram may indicate that more vigorous shaking with
acetone and toluene is required. In products
for which Me Hg recoveries are to be determined, fortify tissue at this point by adding
working fortification solution, to centrifuge
tubes.
Add 2.5 mL HCI solution, to centrifuge
tube containing acetone and toluene washed
sample. Break up tissue with glass stirring
rod, if necessary. Extract CH3HgCI by adding
20 mL toluene and shaking tube gently but
thoroughly 5 mm on mechanical shaker at
setting 5 (2 min by hand). Loosen cap and
centrifuge 5 min at 2000 rpm. If emulsion is
present after centrifugation, add 1 mL isopropanol and gently stir into toluene layer
with glass stirring rod to reduce emulsion.
Do not mix isopropanol with aqueous phase.
Add equal amounts of isopropanol to blank
and test solutions. If emulsion is not present,
do not add isopropanol to blank or test
solutions. Vigorous mixing of isopropanol
with HCI may produce interfering peaks in
chromatograms. Recentrifuge. With dropping pipette, carefully transfer toluene to
graduated cylinder. Rinse walls of centrifuge
tube with 1-2 mL toluene and transfer rinse
to graduated cylinder. Repeat extraction
step 1 more time. Combine both extracts in
graduated cylinder, dilute to 50 mL with
toluene, stopper and mix well. Add 10 g
Na2S04 and mix again. Tightly stoppered
extracts (sealed with Teflon tape) may be refrigerated and held overnight at this point.
Analyse by GC.
4 The Measurement Process in Chemistry
F. Gas Chromatography
Verify that system is operating properly by
injecting 5 p.L standard solution containing
0.005 p.g HglmL into GC system. Difference
between CH3HgCI peak heights for 2 injections should be ~4%. Check detector
linearity by chromatographing all working
standard solutions.
Inject 5 p.L standard solution with concentration approximately equal to or slightly
greater than concentration of extract. Immediately after CH3HgCI peak appears, inject another 5 p.L extract. Immediately after
CH3HgCI and background peaks for extract
appear, inject another 5 p.L aliquot of standard solution. Because column performance
and peak height slowly decrease with time,
calculate Hg concentration in each test
sample by comparing peak height for each
test extract to average peak height for standard solutions injected immediately after test
extract.
Correct height of CH3HgCI peak for test
extract by subtracting height of peak for
method blank obtained at same attenuation
and recorder sensitivity. Calculate Me-bound
Hg content of test sample expressed as p.g
Hg/g (ppm Hg) by comparing height of peak
from injection of test extract to average
height of peak from duplicate injections of
standard solution as follows:
p.gHg/g fish = (RIR') x (e'le) x 50
where
R = corrected height of CH3HgCI peak from
injection of test extract;
R' = average height of CH3HgCl peak from
duplicate injections of standard solution;
e = weight (g) of test portion;
e' = concentration (p.glmL) of Hg in standard solution; and
50 = fmal volume (mL)