dium-phosphotungstate complex into several oxygenated organic solvents. Extraction of the vanadium complex is quantitative a t room temperature with the normal alcohols through dodecanol. With tetradecanol and heavier alcohols, extraction becomes difficult because the extraction mixture must be heated, emulsions difficult to break form at the interface, and the absorbance of the vanadium complex must be determined a t elevated temperatures. Concentrations of chromium(III), cobalt(II), iron(III), nickel(II), and molybdenum(V1) normally found in the high purity aluminum powder used for this work do not interfere in the analysis. Table I11 describes the results obtained by adding the various ions to a known concentration of vanadium prior to formation of the colxed vanadium phosphotungstate complex. I n concentrations 25 times t h t t of vanadium, nickel(I1) does not interfere while iron(II1) interferes only slightly. Chromium(II1) and cobalt(I1) interfere markedly in 25-fold excess and in 1000-fold excess cobalt interference is quite large. Both chromium(II1) and cobalt(I1) interference are eliminated by cathodic reduction. illolybdenum (VI) interferes in 25-fold and greater excess. Cathodic reduction of molyb-
Table 111.
Interference in the Vanadium Determination b y Diverse Ions Added to 0.1 8 Mg. of Vanadium
Absorbance Before cathodic After cathodic Rlg. vanadium Ion Mg. added reduction reduction after reduction Ni(I1) 5.0 0.199 0.196 0.185 0.206 0.194 Cr( 111) 5.0 0.260 Fe(II1) 5.0 0.182 0.196 0.185 5.0 0.183 0.192 0.180 Co(I1) 5.0 0.244 0.199 0.189 200.0 0.750 0.209 0.201 &To(VI)a 5.0 0.312 0 322b 0.312 500.0 0.520 0 . 570h 0.553 a Cathodic reduction not applicable to removal of interference from molybdenum(V1). Solutions hazy and absorbance not representative.
denum does not eliminate the intcrference and has little, if any, effect on it. hIolybdenum(V1) interference is eliminated by extraction of the chloride with diethyl ether. Ordinarily a minimum of five extractions with diethyl ether is required to remove the molybdenum(V1) interference. LITERATURE CITED
(1) Cooner. M. D.. Winter. P. K.. ANAL. \
I
CHI& 21, 605-9 (1949).’ ( 2 ) Hillebrand, W. F., Lundell, G. E. F., Bright, H. A., Hoffman, J. I., “Apphed Inorganic Analysis.” D. 458, Wilev, ” , New-York, 1953:
I
_
(3) Hopps, G. L., Berk, A . h., SAL. CHEW24, 1050-1 (1952). ( 4 ) Majumdar, S. K., De, A. IC., Ibid., 33, 297-9 (1961). (5) Milner, 0. I., Glass, J. R., Kirchner, J. P., Yurick, A. X., Ibid., 24, 1728-32 (1952). (6) Morrison, G. H., Freiser, €I., “Solvent Extraction in Analytical Chemistry,” pp. 143-4, Wiley, New York, 1057. ( 7 ) Pearse, G. A., Jr., ASAL. CHEM. 34, 536-7 (1962). (8) Ryan, D. E., d n a l @ 85, 569-74 11960). (9jWright, E. R., Slellon, 11. G., IND. E K G . CHEM., ANAL.En. 9, 251 (1937). RECEIVEDfor review -4pril 1, 1963. Accepted July 10, 1963.
Fluorometric Determination of Selenium in Plants and Animals with 3,3’-Diaminobenzidine W. B. DYE, ERICH BRIITTHAUER,’ H. J. SEIM,2 and CLIFTON BLINCOE University of Nevada, University Station, Reno, Nev.
b Two methods of oxidizing samples (Parr bomb and Schoniger flask) for the fluorometric determination of selenium in plants and aniinals have been developed and/or improved. Intensive and detailed studies have been made on the variables affecting the results. These methocls are superior to previous methods in that only a small sample i s required; extraneous fluorescence i s more efficiently removed; the time of analysis i s materially decreased. The combustion techniques, measured b y 1 3 determinations with each technique on the same ground sample o f alfalfa, gave for the Parr bomb an average o f 1.1 4 p.p.m. of Se with a s’tandard deviation of A0.019; for the Schoniger flask, an average o f 1.17 p.p.rn. of Se with a standarcl deviation of A0.012. Results obtained b y the fluorometric 3,3‘-d i a minob enzidine (DAB) method agree reasonably well with
neutron activation determinations. The fluorometric DAB method has been tested on alfalfa, grass, linseed meal, wheat, corn, oats, lettuce, and pear, apple, and radish leaves; and on blood, muscle, liver, brain, and milk.
F
many years the interest of research workers has been directed toward the investigation of selenium (Se) toxicity in farm snimals. Forage containing Se in exec‘s of 5 p.p.m. is toxic to cattle. Recently investigators (3-6, 8, 12) have shown that a Se deficiency may exist in cattle and sheep; muscular dystrophy (white muscle disease) may occur if the feed ingested is lorn in Se. Furthermore, sheep are unthrifty on low Se diets even if white muscle diseaie does not occur (8). Conventionally the analyst may uw the .LO-AC (1) method of determination OR
in plant material exceeding 2 p.p.m. of Se. The ground plant sample is mixed with H2S04and HKOa in a beaker and after digestion the digest is transferred to a distillation flask. The digest is distilled with HBr and Brz and the distillate collected in a cooled Erlenmeyer flask. SO2 is passed into the flask to remove excess Brg, and hydroxylamine added to reduce selenious acid to elemental Se. The precipitated Se is measured either gravimetriraily or colorimetrically. There are serious objections to thi.: method, particularly when plant or animal samples are analyzed for Se. The method requires .i grams of plant sample; it has a relatively low sensitivity, since it is applicable only to Present address, Southwestern Radiological Health Laboratory, Las Vegas, Sev. * Present address, Allis Chalmers l l f g . Co., Milwaukee 1, Wis. VOL. 35,
NO. 1 1 , OCTOBER 1963
1687
Abridged Comparison of Cousins' (3) and Watkinson's (17) Methods for Se in Biological Material
Table 1.
stcp 1 2
3
4 5
0
7
8 9
I (Cousins) Drying sample Plant tissue, yes Animal tissues, usually not Grinding (for plants) Weighing, 1 to 10 grams of animal tissue Oxidation, HNOZ HClOd digestion Isolation of Se a. Reduction to Se(IV) b. Coprecipitation of Se with As e. Filtration d. Digestion (HN03 HCIO,) Complexation (HCOOH EDTA) Reaction with 3,3'-diaminobenzidine (DAB) Extraction (toluene) Measure fluorescence
+
+ +
Table II.
stcp 1
2
3
4
)
6
7 8
9 10 11
12 13
14 15
1688
XI (Watkinson) Dry at 30°-400 C. (plant tissue) Plant tissue, finely ground Amount of material to give not less than 0.02 pg. Se Same Isolation of Se Reduction to Se(1V) (HC1) Complexation of Se (zinc dithiol) Extraction (C,H&lz CClr) Digestion (HNOz HCIO,) Same Same
+
Same Same
Fluorometric Determination of Se in Plant Material
Parr Bomb Grind air-dried plant sample (80mesh screen) Pellet and weight accurately 0.10- to 0.20-gram sample into fused silica ignition capsule Attach platinum fuse wire and nylon yarn (16). Add 10 to 15 ml. H20 as absorbing liquid and assemble bomb. Use 30 atm. of oxygen and ignite as per instructions (16). Cool bomb in tap water for 10 minutes. Release excess oxygen over a period of 2 minutes Rash contents of bomb into 150-ml. beaker, using small portions of H2O from wash bottle Filter mlution through S & S white ribbon paper into 250-ml. beaker Make filtrate (about 30 to 35 ml.) approximately 4iV by adding 10 ml. of concd. HC1 Boil for 10 minutes t o reduce all Sc to Se(1V) state Add 1 ml. of concd. HCOOfI Add 5 ml. of 0.1111 EDTA (dimdium salt) Add 1 ml. of 0.5"1, 3,3'-disminobenzidine hydrochloride solution (freshly made) Adjust pH 2.0-2.4 (NH40H) Set in subdued light 30 minutes Adjust pH with X&OH to 0.8-7.2. Make solution volume to 50 ml. with €I& Extract (VirTis) with 5.00 ml. of toluene (or mesitylene for low concentrations of S e ) Read fluorescence of Turner fluorometer using filters 2.4 47B for primary filter nud 2-,4-12 for secondary filter. Note sensitivity and range as wc~llas dial reading. For extremely low concentrations of Se, use Turner high sensitivity kit
+
ANALYTICAL CHEMISTRY
+
by Method 111
Schoniger Flask Same Weigh accurately 2 p.p.m. of Se; it is very laborious and time consuming. Hoste (9) and Hoste and Gillis (10) introduced 3,3'-diaminobenzidine (DAB) as a selective colorimetric reagent for selenium. The piazselenol or selenium-diaminobenzidine complex (Se-DAB) formed when Se(1V) reacts with 3,3'-diaminobenzidine or its hydrochloride has an intense yellow color. Se-DAB can be readily extracted from a slightly acid or neutral solution and measured colorimetrically. Cheng (2) and Parker and Harvey (16) have studied the chemistry of this reaction; Cheng reported the limit of sensitivity to be 0.05 p.p.m. of Se using a 1-cm. absorption cell. Handley and Johnson ( 7 ) have used the same reaction with plant material and report that 0.25 pg. of Se may probably be determined with confidence. hIagin et al. (14) use this method for measuring Se in water. Cousins has been credited with discovering that the piazselenol fluoresces. Cousins (3, 4) and Watkinson (17) have developed fluorometric methods (Methods I and 11) for measuring low concentrations of Se in biological material. An abridged comparison of these methods is shown in Table I. The Se-DAB complex formed by the reaction of Se(1V) with 3,3'-diaminobenzidine or its hydrochloride, when activated with light a t wavelengths of 420 to 430 mp, fluoresces a t wavelengths of 560 t o 585 mp; although the blank has a rather high fluorescence, the fluorometric method is more sensitive than the colorimetric method. Methods I and I1 are still laborious and time-consuming and subject t o extraneous fluorescence which some workers ascribe to lipides or lipoidal material in the sample. It also seems likely that in a plant tissue sample much of the extraneous fluorescence is due t o unoxidized plant sample, or, rather, unoxidized chlorophyll and /or xanthophyll; both of these plant pigments are known to fluoresce strongly. This extraneous fluorescence following HKOa or HX0a+HCiO4 treatment apparently cannot be removed by orgniiic extraction; neither can it be removed by adsorption using the common ndsorbents such as charcoal, Darco G-60, or diatomaceous earth. Cousins (3) avoids this difficulty by isolating Se by coprecipitation with As; Watkinson ( 1 7 ) separates Se from a plant digest by complexing the Se with zinc dithiol and extracting. The improvement of proposed Methods I I I a (Parr bomb) and I I I b (Schoniger flask) over the methods of Cousins (3, 4) and Watkinson ( I T ) is due to two factors: 1. A small sample (0.10 to 0.20 gram) of ground plant tissue is oxidized in a 2-liter Schbnieer flask or a Parr bomb. This is much more convenient
120-
100-
>
t
g 80c w E w 60-
r
s 40 e
WAVELENGTH,
WAVELENGTH,
m~
Figure 1. Activation !;pectra on Aminco-Bowman spectrophotofluorometer
Figure 2. Fluorescence spectra spectrophotofluorometer
- Se(lV)-DAB fluorescence setting, 565 m p --Reagent blank fluorescence setting, 565
mp
and rapid than oxidizing or digesting a larger plant sample ir, acid. For very low concentrations of Se in plants (less than 0.10 p.p.m.), three methods are readily available to in1:rease the magnitude of the fluorometer readings. If only an occasional l o r Se measurement is required, a second sample may be oxidized in the Parr bomb or Schoniger flask after cooling the previous sample; it is not necessary to wash out the bomb or flask contents between ignitions. If a series of low Se measurements is required, the Turner high sensitivity kit attachment can bc used to increase the sensitivity of the fluorometer by a factor ranging from ii to 10. Gutenmann and Lisk (6) have also used a 5liter Schoniger flask (Tnomas-Lisk style) with a larger sample for the drtermination of Se in plants. Figure 10 shows t h a t the use of mesi1)ylene instead of toluene as the extractant will also increase the sensitivity 0:'the method. 2. The weight of unburned plant material particles of Methods I I I a and b ranges from 0.1 to 0 2 mg. per determination, which is an insignificant amount of the sample, these unburned particles are filtered off immediately and contribute no extraneous fluorescence from their chlorophyll and/or other plant pigment content. This simple method remove's the extraneous fluorescence easily and effectively. EXPERIMENTAL
Apparatus. Parr bomb. Fused silica capsules (Parr List No. 4383) with platinum wire (26-gauge) and nylcn varn (16). Schoniger flask, lhoma&Lisk FtyIP, 2-liter capacity. Spectropliotofluorometer, Aminco-Bowman. Fluoronieter, T i m e r Model 110, with and without hig'i sensitivity kit. Fifttmi-millimt tar ci,vettcs. VirTis IC.;trwtomrLtic. with IOO-mI. tulm and 'I'eflon stopcxoc-ks. pII meter, lkckmxn Zerornatic. Scintillation counting assembly, Xiiclear-Chicago scaler (hIodel I S M ) and pulse-height analyzer (Model
1810) or a Technical Associates Scaler (Model 1)s-58) and pulse-height analyzer (Model SA-20) utilizing a thallium-activated sodium iodide crystal. Homogenizer, VirTis or equal. Lyophilization apparatus: a desiccator connected to a Pirani gauge and a vacuum pump through a cold trap is adequate. Reagents. All reagents were of analytical grade and included NHIOH, HCl, "Os, toluene, mesitylene, formic acid (88 t o 90%), EDTA (disodium salt), and 3,3'-diaminobenzidine hydrochloride (J. T. Baker). Water redistilled from glass was used in making up all solutions, since i t contained less fluorescent impurities than distilled, deionized (mixed bed) water. AI1 glassware was washed with a nonfluorescing detergent solution and rinsed with distilled water, followed by rinsing in redistilled water and oven drying. Care was taken to keep fingerprints off cuvettes. Standardization. A selenium(1V) standard stock solution was prepared by dissolving 0.500 gram of elemental Se (99.9%) in a few drops of " 0 3 and diluting with 4N HCl to 1 liter. This 500 p.p.m. Se(1V) standard stock solution was further diluted with 4N HCl to prepare standard solutions of 50, 5, 0.5, 0.05, and 0.005 p.p.m. of Se. From these dilute standard solutions, aliquots were taken to make standard curve A shown in Figures 3 and 4. Air (oxygen) will slowly oxidize Se(1V) to Se(V1) unless the solutions are boiled with 4N HC1 or prepared with 4N HCl. Se(V1) reacts with DAB to produce a compound with greatly decreased fluorescence when activated a t 420 to 430 mp. The detailed steps for making the standard curves (Figures 3 and 4) are shown by steps 7 to 15 of Table 11. Figure 1 shows the activation curve obtained with the Aniinco-Bowman spectrophotofluorometer for the DAB blank and Se(1V)-DAB complex. The activation peak of Se-DAB is a t 425 mp; Figure 2 shows the fluorescent curve taken with the same instrument. The
m~
on Aminco-Bowman
Se(iV)-DAB activation setting, 425 m p Reagent blank activation setting, 425 m p
fluorescent peak of the Se(IV)-DAB complex is a t 565 mp. The standard curves shown in Figures 3 and 4 were made using the Turner fluorometer. The primary filters used were a sharp-cutoff 2A filter plus a narrow-pass 47B filter; the secondary filter was 2A-12 sharp-cutoff filter (designations are Wratten numbers). Figures 3 and 4 show standard working curve A to be linear up to a t least 0.20 pg. of Se per 5 ml. of toluene. The standard working curve is adequate for sample weights of alfalfa ranging from 0.10 to 0.20 gram. PROCEDURE
Plant Samples. Representative airdried samples were first ground in a standard model Wiley mill (40-mesh screen) and a small subsample was taken and reground in a n intermediate Wiley mill (80-mesh screen). Detailed steps using the Parr bomb (Method IIIa) and the Schoniger flask (Method IIIb) are shown in Table 11. Safety precautions for both ignition procedures included: use of small samples in the bomb or flask; a balloon attached to Schoniger flask; all flask ignitions made behind R plastic safety shield. The fluorescence measurements for plant sample solutions and standard solutions were made in exactly the same manner. Animal Samples. BLOOD. Parr Bomb. Adsorb 1 ml. of serum on a 11-cm. ashless filter paper. Lyophilize (or dry) the sample to dryness (approximately 4 hours). Cut paper into small squares (approximately I/* inch) and place in silica ignition capsule. Alternately the paper may be pellebed after cutting. Follow the procedure of IIIa beginning with step 3. Schoniger Flask. Adsorb 1-nil. serum sample as for Parr bomb. Fold the filter paper into a square (approxiniatelys',1 inch,. lnsert Srliiiniger wick (niatlr from Schoniger ignition paper) into folded somple. Follow procedure of Rlethod IIIb beginning with step 3. MILK. Same as serum. V O I 35, NO. 11, OCTOBER 1963
1689
B 004
002
006
008
0 0
n,
V
0
l
012
014
016
018
q
020
&a S s / 5 ml TOLUENE
Figure
,qg
3. Effect of valence of Se on standard curves
Se/5
ml TOCUENE
Figure 4. Effect of reduction methods on fluorescence of Se-DAB complex
A.
Se(lV) 6. Se(VI) Range 30X; sensitivity 3
A. SellV) 6. SelVI) reduced with HCl C. Se(Vl) reduced with NH2OH Range 30X; sensitivity 3
‘I’rssuc. Homogenize a t least 2 grain5 of sample. Spread sample in a thin layer on a planchet and lyophilize (12 t o 20 hours). Powder the sample, using a small mortar and pestle. Weigh out 0.1 to 0.2 gram of pulverized sample into Schimger ignition paper. Follom the procedure of Method IIIb, beginning ITith step 3. STUDY O F VARIABLES AFFECTING FLUORESCENCE O F Se-DAB
An extended and detailed study was made of all variables which might reasonably affect the fluorescence of Se-DAB solutions. I n general, standard solutions of Se were used; the reagents and their concentrations were the same as previous workers had found to be satisfactory. Figures 3 to 10 show the effects of these variables, and since the Turner fluorometer was used for this study the ordinate of each curve ib expresbcd as the dial reading a t a designated range for this instrument. The dial reading is proportional to the fluorescence of the Se(1V)-DAB toluene extract. Valence. Selenium(1V)-DAB has a maximum activation peak a t approximately 425 mp (Figure l), and a
inaxinium fluorebcence peak a t approximately 566 nip (Figure 2), which are suitable for fluorescence mwurements. Se(V1)-DAB does not activate or fluoresce a t these wavelengths. It is necessary to ensure that all the selenium is in the +4 valence state; otherwise the results of an analysis will be too low, as shown by standard curve B in Figure 3. If sufficient time is permitted to elapse, standard solutions of Se(1V) will slowly change to &(VI) because of atmospheric oxygen. This is not surprising, since it can be shown thermodynamically using the data of Latimer ( I S ) that:
O2 + 2Hae03 = 2 S e 0 a - 2 E,
=
+
4H+ +0.08 volt
This raction, although slow, proceeds as written in Equation 1. I t should be possible to study the rate of this reaction by using a spectrophotofluorometer, or a fluorometer with the proper filters. Reduction Methods. The selenium solution obtained in steps 6 and 7 should be a t least 4s in HCI and boiled approximately 10 minutes.
t
1
(01
i
0o
2
\
60
(1)
Figure 4 (curves A and B ) indicated good agreement for the fluorescence results when two standard solutions, Se(1V) and Se(VI), are so treated. Hydroxylamine is too strong a reducing agent for this reaction; some of the Se(V1) is converted to elemental Se. Varying pH before Development. Figure 5 shows the fluorescence results when the p H is varied immediately following the addition of DAB. A pH of 2.0 to 2.4 (step 11) is satisfactory, as was also noted by Cheng ( 2 ) ) who was studying the colorimetric measurement of Se-DAB. Varying pH before Extraction. The fluorescence curve obtained in Figure 6 also corroborates fluorometrically the colorimetric results of Cheng ( 2 ) . A pH of 7 is therefore used in step 13. Varying Reaction Time before Extraction. The effect on fluorescence of varying reaction time of Se(IV) with DAB is shown in Figure 7 . The solution was kept in subdued light a t room temperature (23’ to 24’ C.). The curve shows t h a t a time of 30 minutes is adequate for step 12.
i
Y
3
0
L
-
-
~
-
-
-
-
1
-
-
_
_
-
~
1 3
?H
I.-___d----i ?H
Figure 5. Effect of varying pH immediately following addition of DAB on fluorescence of Se-DAB complex
Figure 6. Effect of varying pH of aqueous phase before extraction, on fluorescence of Se-DAB complex
Range 30X; sensitivity 3
Range 30X; sensttivity 3
1690
ANALYTICAL CHEMISTRY
TIME,
10
MINUTES
all steps following step 11 should be made in subdued light, or when possible, in darkness; fluorescence of Se-DAB in toluene begins t o decrease after standing 30 to 35 minutes even in subdued light. Temperature during Standing Time. Figure 9 illustrates t h e effect
of
fluorometer dial readings* 0 0 0 0 0 0 0 0 0 0 78 0 0 0 0
2,000 30,000 c11,000 Cu(I1) 1,000 Ce(111) 1,000 Co(IT1) 100 CrlII) 1.000
FeiIIj I;OOO Fe( 111) 1,000 Fe(I1) 10,000 K(1) 1,000 Mn(I1) 4,000 Mo(1V) 1,600 Mo(1V) 1,000 Tali)
Ni(I1) Pb(I1)
1.000
1;OOO 1,000
SilTV',
i.nnn
Te(1V) Te( I V )
1:ooo 2,000 (Te metal) 2,000 ( T e c h )
kn(1V) Te(I\ ) Te(I?) Te(l\) V(I1) Y(II1) K(I1) Zn(I1)
2,000 (Te02,Baker) 1 ,000 ( Te02, Baker) 100
(TeOz. Baker)
0 0 0 0 0 98 42 26 14 0
1,000 1,000 2,000 1,000 r o 3 1,000 PO-3 1,000 so4-z 1,000 0 TOBO-ml. aliquot of standard solution containing 0.05 pg. of selenium. This solution has a fluorometer dial reading of 12.5 divisions. b Range 30X ; sensitivity 3 on Turner Q
fluorometer.
10
80
Subduedlight 6. Sunlight Range 30X; sensitivity 3
Increase
As(V) Ca(11)
60
W
A.
Table 111. Effect of Civerse Inorganic Ions on Fluorescence of Se-Dab Complex
In Amount organic added,a ion (rg.) Al(II1) 1,000
40
Figure 8. Effect of standing time on fluorescence, after extraction of Se-DAB complex
:$OX; sensitivity 3
Standing Time in Subdued Light of Extracted Se-DAIB. A toluene solution containing Se-DAB was divided into two parts: Solution A kept in subdued light (semidarkened room) remained more fluorescent t h a n Solution B in sunlight, :is indicated in Figure 8. These results indicate t h a t :
30
TIME AFTER EXTRACTION, MINUTES
Figure 7. Effect of varying reaction time of Se(lV) with DAB (in subdued light), on fluorescence of Se-DAB complex Range
20
o
60 L
l
T I M EI20 , UINUTES
I80
Figure 9. Effects of temperature and standing time (in darkness) on toluene extracts of Se-DAB
on fluorescence of varying the temperature of Se-DAB toluene extracts kept a t constant temperatures in darkness. The fluorescence decreases with temperature; in this experiment, a rise of 1' C. reduced the measurement of Se by 0.0084 p.p.m. K h e n measuring small amounts of Se, it is advisable to let the toluene extracts stand in an oven or a constant temperature room a t the specified temperature. Varying Extractants. Figure 10 shows the fluorescence results obtained with standard solutions using five different extractants. Mesitylene appears t o be the most favorable; however, toluene is cheaper and more easily obtained; for most purposes a good grade toluene is satisfactory. Inorganic Ions. The effect of diverse inorganic ions on fluorescence iq shown in Table 111. The samples of T e and its salts probably had Se as a n impurity, judging from the erratic results. If a large amount of iron is present in a sample, more E D T A should be
used.
40
vr
P 30
n A
2
g 20 c
E 5
2
1 0
0 .qg
S e 1 5 ml E X T R A C T A N T
Figure 10. Effect of various extractants on standard Se-DAB fluorescence A.
Mesitylene 6. Toluene C. o-Xylene D. p-Xylene E. Benzene
VOL. 35,
NO.
1 1 , OCTOBER 1963
1691
RECOVERY EXPERIMENTS
Radiotracer methods were used in checking the recoveries of a number of different steps in Method 111(Tables IV, V, and VI). Alfalfa was grown in the greenhouse using a modified Hoagland macronutrient solution with micronutrient concentrations as recommended by Johnson (11). Each 19-liter nutrient solution contained 100 pc. of Se76(3000 pc, of Se76 per gram of Se). The to l/, alfalfa w w harvested at bloom stage. Grinding and other analytical steps were according t o steps in Method 111. Radiotracer measurements were made, preceding and following each step or experiment. Total re-
Table IV. Radiochemical Recoveries of Se-75 Using Various Absorption Media
Absorbing media H20 0 . W NaOH 0.1N HCl Ha0 0. IN NaOH 0.1N HCl
Oxidation Schoniger Parr bomb
Recovery,
%
100 f 1' 100 f 10 100 f lo 100 f l a 100 f 1' loo f la
Relative standard deviation.
Table V. Recovery of Se-75 Using Various Organic extract ants^
Solvent Benzene Toluene o-Xylene en:& :% ;! Cumene Bromobenzene Chlorobenzene Nitrobenzene o-Nitrotoluene m-Nitrotoluene Chloronaphthalene Nitrobiphenyl Cyclohexane C clochlorohexane ethylcy clohexene Hexane Chlorohexane Carbon tetrachloride Chloroform Bromoform Trichloroet,hane Ethylene chloride Chloromethylbutane Isoam 1 alcohol Capryic alcohol Octanol Decyl alcohol Diethyl carbinol Butyl acetate Amy1 acetate 3-Heptanone
d
Selenium-75 recovery, % 77.8 f 3 6 73.8 f 3 69.9 f 3 70.1 f 3 80.0 f 3 66.1 f 3 56.2 f 3 69.2 f 3 79.0 f 3 92.0 f 3 76.2 f 3 77.5 f 3 74.4 f 3 25.2 f 3 83.0 f 3 20.0 f 3 0.5 f 3 34.1 f 3 45.8 f 3 84.5 f 3 41.3 f 3 71.0 f 3 86.7 f 3 22.1 f 3 80.0 f 3 80.0 f 3 82.5 f 3 61.3 f 3 69.0 i 3 73.6 f 3 75.4 f 3 80.4 i 3
covery calculations are based on the assumption that total Se is recovered to the same per cent as Se75. Use of Various Absorption Media. Radiotracer recoveries were made on 0.10 t o 0.12 gram of alfalfa containing Sei6 after combustion in a 2liter Schoniger flask using various liquid media. Table IV shows that the recovery from the flask is complete; water, 0.1N HCI, and 0.1N XaOH absorb equally well; and 25 ml. of liquid is sufficient. Water was chosen for the absorbent, because the absorbing solution after combustion is acid and the p H has t o be raised. The Parr bomb gave similar results, and required only 10 t o 15 ml. of HzO. Extraction Results. A stock s o h tion of Se(1V) containing some Sei6 was made u p and a n aliquot taken for fluorometric analysis (steps 6 t o 15, Method 111). Radiotracer measurements were made before and after extraction with toluene. The results for 50 ml. of aqueous phase and 5 ml. of toluene indicated that a recovery of 74 f27, would be obtained with one extraction; there is no advantage ( I C ) in attempting to increase the per cent extracted by increasing the number of extractions, because the volume of the organic phase will also be increased. Table V shows the radiotracer recovery results for various organic extractants. It, appears that many organic extractants are superior to toluene for extracting Se from aqueous Se-D.4R solutions; unfortunately, Se-DrlB does not fluoresce in many of these organic extractants. An exception is mesitylene (Figure 10). Plant Samples. Radiotracer recoveries were run on alfalfa samples using Methods IIIa and I I I b (Table VI). Slightly better precision is obtained with the Schoniger flask; there is little to choose between the Parr bomb and the Schoniger flask with respect to the average value. The bomb will oxidize larger samples than a 2-liter flask, but the cost of the bomb is greater. More samples can be oxidized per day with two flasks than with one bomb.
Table VIII.
Plant tissue Pear leaves Apple leaves Radish leaves Lettuce leaves Corn
Wheat Sample 1, Alfalfa'
b
Relative standard deviation.
1692
ANALYTICAL CHEMISTRY
b
0
Comparison of Analytical Methods. hlethods IIIa and I I I b were compared by analyzing 13 replicates of a ground alfalfa sample by each method. T h e
Table
VI.
Fluorometric Determination
of Se in Plant Material. Recovery Experiment Method I11 Sei6 recovery through individual Step steps, 5% 1. Air-dry sample ... 2. Grind (60-mesh screen) 3. Weighing 100 * 2 b a. Parr bomb (0.10 . 2 gram) b. Schoniger flmk 1
