9.2.

30
AOAC Official Method 974.15
Selenium in Human and Pet Food
Fluorometric Method
First Action 1974
Final Action 1976
A. Apparatus
( a ) F l u o r o m e t e r . F i l t e r f l u o r o m e t e r o r
spectrophotofluorometer capable of excitation at 366 nmand detec-
tion of fluorescence at 525 nm.
(b) Cuvets or tubes.Pyrex culture tubes, 12 75 mm, selected
by matching, are suitable for fluorometer.
(c) Wrist-action shaker.Model BB (Burrell Corp.), or equiva-
lent, set at maximum speed.
(d) Separators.Glass, 250and125mL, withTeflonstopcocks.
B. Reagents
(Use analytical grade reagents and glass-distilled H
2
Othroughout
except as noted.)
(a) Nitric acid.Distil fromglass, discardingfirst andfinal 10%.
(b) Dilute sulfuric acid.2.5M. Dilute 140 mL H
2
SO
4
to 1 L with
H
2
O.
(c) Ammonium hydroxide solution.Approximately 6M. Dilute
400 mL NH
4
OH to 1 L with H
2
O.
(d) Disodium EDTA solution.0.02M. Dissolve 7.445 g
Na
2
H
2
EDTA2H
2
O and dilute to 1 L with H
2
O.
(e) 2,3-Diaminonaphthalene (DAN) solution.1 mg/mL. Pul-
verize DAN(purest grade available; product fromAldrich Chemical
Co., Inc., No. 13.653-0 has been found satisfactory) in clean mortar
to fine powder. Insert glass wool plug in stem of 250 mL separator
and add 150 mL 2.5M H
2
SO
4
. Transfer 0.150 g DAN to separator
and place on shaker 15 min to dissolve. Add 50 mLcyclohexane and
shake 5 min. Let phases separate 5 min, drain lower phase into an-
other separator, and discard cyclohexane (upper) phase. Repeat cyc-
lohexane extraction twice more; after third extraction, drain lower
phase into low-actinic glass-stoppered flask, add 1 cmlayer hexane,
and store in cold. Solution is stable several weeks.
( f ) Sel eni um s t andar d s ol ut i on. ( 1) St ock s ol u-
tion.100g/mL. Dissolve 0.1000 g black Se (purity 99.9%) in ca
5 mL HNO
3
, (a), and warm to dissolve. Dilute with H
2
O and 20 mL
2.5M H
2
SO
4
to 1 L. (2) Working solution.Dilute stock solution
with H
2
O and 2.5M H
2
SO
4
to give Se concentrations in 0.05M
H
2
SO
4
appropriate for level of Se expected in material. Store all so-
lutions in all-glass containers. Solutions are stable indefinitely.
C. Preparation of Standard Curve and Fluorometric Blank
Conduct appropriate volumes of Se standard solutions (10 mL
containing 800 ng Se) and 10 mLH
2
Oeach through entire determi-
nation, including digestion, along with samples. Zero fluorometer
against blank solution and read fluorescence at 525 nm or subtract
blank fluorescence from that of standards. Plot reading against ng
Se/6 mL cyclohexane solution. Prepare new standard curve daily.
D. Determination
(Toensure adequate cleanliness for fluorometry, acid-washall glass-
ware except cells. In particular, clean Kjeldahl flasks and Erlenmeyers,
separators, centrifuge tubes, andglass beads before eachdetermination.
Rinse glassware with hot H
2
O, dry in oven, and wash with hot
HNO
3
H
2
SO
4
[1 + 1]. Rinse with hot tap H
2
O followed by distilled
H
2
Oand dry in oven or let air dry. Rinse cells with alcohol followed by
acetone. Do not use plasticware other than that mentioned.)
Place accurately weighed test portion containing 1.0 g dry mat-
ter and 0.8 g Se with 3 glass beads into 100 mL Kjeldahl flask
containing 10 mLH
2
O, and swirl to wet sample. Add 10 mLHNO
3
,
B(a). (Alternatively, omit the 10 mL H
2
O, add 10 mL HNO
3
, or
more if all HNO
3
is absorbed by sample, and let digest overnight at
room temperature.) Heat cautiously to reduce volume to ca 5 mL,
taking care to prevent severe foaming or bumping, and cool. Add
6.0 mL 70% HClO
4
and 5.0 mL H
2
SO
4
, return to cool heater, and
heat until solution first turns yellow and then becomes colorless.
Avoid charring of sample during digestion which may result in loss
of Se. If charring occurs, repeat analysis with new test portion, us-
ing higher HNO
3
HClO
4
/test portion weight ratio. If this fails, add
small amounts of HNO
3
at first signs of darkening.
Remove flask from heat, swirl to wet entire bulb area and lower
neck of flask, replace flask on heater, and continue heating until so-
lution becomes colorless and white fumes appear.
Remove flask from heat, swirl, add 1.0 mL 30% H
2
O
2
, rinsing
walls of flask, and swirl until fuming ceases. Resume heating until
contents boil briskly and white fumes are again evolved. Repeat ad-
dition of H
2
O
2
and heating twice more, and continue final heating
5 min after appearance of white fumes. Let flask cool, add 30 mL
H
2
O, rinsing walls of flask, and mix thoroughly. Transfer quantita-
tively to 250 mL glass-stoppered Erlenmeyer, using two 10 mL and
one 5 mL H
2
O rinses. Add, successively with mixing, 10.0 mL
EDTA solution, 25.0 mL 6M NH
4
OH, and 5.0 mL DAN solution.
Bring quickly to brisk boil and boil exactly 2 min.
Let reaction mixture stand at room temperature for definite inter-
val between 1 and 2 h. Use same interval for all samples, standards,
and blank in set. Accurately add 6.0 mL cyclohexane, stopper flask,
and place on shaker 5 min. Transfer to 125 mL separator, and let
phases separate ca 5 min. Discard lower aqueous phase and drain
cyclohexane solution into 15 mL centrifuge tube. Centrifuge 5 min
to further separate H
2
O and transfer ca 5 mL to fluorometer cell.
Zero fluorometer against reagent blank and read fluorescence of
test solution at 525 nm. Alternatively, subtract fluorescence of blank
fromthat of test solution. Determine Se content fromstandard curve.
Although fluorescence readings for both test solutions and blanks
increase with time, net readings (test solution blank) remain
consistant with 12 h complexing period.
Reference: JAOAC 57, 368, 373(1974).
CAS-7782-49-2 (selenium)
Revised: March 1996
2000 AOAC INTERNATIONAL