chimney is comfortably warm to the touch. When most of the nitric acid has evaporated, 1.0 ml. of 70% perchloric acid is added and heating is continued until white fumes appear and the digest appears colorless. An additional 15 minutes’ heating ensures complete digestion, which is essential. Two milliliters of glass-distilled water followed by 15 ml. of hydrobromic acid-bromine mixture are added to the flask via the chimney, which is thoroughly washed thereby. The chimney is then removed and the flask clamped to the distillation apparatus. A 100-ml. beaker containing 1 ml. of glass-distilled water is placed beneath the delivery tube and tilted so that the tip of the tube is immersed. The side arm of the boiling flask is then connected to a nitrogen supply and a fairly rapid flow of nitrogen is passed through the apparatus. Heating is then begun, using a microburner protected from drafts and an asbestos pad. The distillation must be carried out in a fume hood because bromine is evolved. During distillation the flame is adjusted so that only a very small amount of hydrobromic acid distills over, the descending (short) limb of the condenser remaining cool. After l l / z hours, heating is discontinued and, after cooling, the boiling flask is removed. The condenser and delivery tube are then washed with a total of 9 ml. of glass-distilled water and the washings transferred to the collecting beaker. Two drops of phenol solution (5%) are added to the distillate to destrov any residual bromine and the pH i”s adjusted to about 7.0 with concentrated ammonium hydroxide. Formic acid (88 to 90%) is added dropwise to a p H of exactly 2.5. Two milliliters of the diaminobenzidine solution are then added. This volume is not critical; variations between 0.5 and 5.0 ml. did not affect absorbance a t 420 mp after toluene extraction. After an hour the pH is again made neutral with ammonia.
Diluted ammonia (1to 5) is useful in the latter part of this adjustment, because the formic acid-ammonia mixture is not well buffered near neutrality. After neutralization, the solution is transferred to a small separatory funnel and the volume adjusted to 25.0 ml. Although not used for this work, cylindrical graduated funnels should be excellent for this purpose. One milliliter of toluene is then added and the mixture is vigorously shaken for 10 minutes on a mechanical shaker. Finally, the toluene is drawn off and centrifuged briefly and its absorbance is determined a t 420 mp using a microcuvette with a capacity of 0.7 ml. The absorbance should be determined as soon as possible, because the toluene solutions darken slowly, probably because of the oxidation of unreacted diaminobenzidine. EXPERIMENTAL RESULTS
The relationship between selenium content and absorbance is shown in Table I. These data were obtained by distilling standard solutions prepared as previously indicated. Direct analysis of these standards without distillation was unsatisfactory because part of the selenium is present as selenate which does not react with diaminobenzidine. Significant departures from Beer’s law occurred when the absorbance measured exceeded 1.0. Above this level appropriate dilution with toluene is necessary to preserve linearity. A plot of the data in Table I suggests that values are slightly low in the 0- to 5-7 range. Therefore, in some applications, it may be important to prepare calibration curves over this range. The data in Table I1 were obtained by adding the indicated amounts of selenium to 1.0-gram samples of plant
material and carrying out the whole procedure. Reproducibility is apparently satisfactory and amounts of selenium as low as 0.25 y may probably be determined with confidence. This method is probably no more time-consuming than most chemical methods for this element and does not require expensive equipment, as do x-ray and other physical methods. The amount of material which may be analyzed is limited cnly by the size of the boiling flask used. Where larger quantities of sulfuric and perchloric acids are used, however, it is important to add an equal volume of water before distillation to prevent oxidation of bromine by high concentrations of acid. The precision and sensitivity of this method recommend its use in studies of selenium essentially and in other fields such as mechanisms of salt absorption by plant roots. LITERATURE CITED
(1) Brownlee, K. -i.,“Industrial Experi-
mentation,,’ Chemical Publishing Co., Brooklyn, N. Y., 1949. ( 2 ) Cheng, K. L., ANAL.CHEM.28, 1738 (1956). (3) Handley, R., Stout, P. R., Johnson, C. hI., this laboratory, unpublished data. (4)Robinson, W. O., Dudley, H. C., Williams, K. T., Byers, H. G., IND. ENG.CHEM.,ANAL.ED.6,274 (1934). (5) Trelease, S. F., Beath, 0. A., “Selenium,’, pp. 253-62, pub. by authors, New York, 1949. (6) Williams, K. T., Lakin, H. W., IND. ENG. CHEW.,ANAL.ED. 7, 409 (1935).
RAYMOND HANDLEY C. 31.JOHNSON University of California Berkeley 4, Calif.
Colorimetric Determination of Selenium in Biological Materials SIR: The refinement by Cheng (1) of the colorimetric method of Hoste and Gillis (3) makes possible the quantitative estimation of selenite with an accuracy not previously attainable. Coupling of a method for digesting organic or biological samples with the Cheng procedure widens its range of application. In our hands, the most satisfactory coupling procedure is one involving precipitation of selenium from the solution formed by the usual nitricsulfuric acid digestion (4) of the sample. This is made possible by the addition of 6M hydrochloric acid to the sulfuric acid solution remaining after digestion. The chloride ion forms a sufficiently stable complex with iron to prevent interference by the quantities of this element 2106
ANALYTICAL CHEMISTRY
found in blood and tissue samples and eliminates interference by sulfate. EXPERIMENTAL
Dissolve samples ranging to 10 grams of dry matter or 30 to 50 grams of wet tissue, milk, blood, etc., in 25 ml. of sulfuric acid to which have been added 25 ml. of concentrated nitric and about 0.1 gram of mercuric oxide. Heat in Kjeldahi flasks connected to a manifold, adding 5- to 10-ml. portions of nitric acid if darkening occurs, until a temperature of 180” to 190” C. can be maintained for a few minutes without darkening. Rinse sample into a 250-ml. Erlenmeyer flask with 25 ml. of water, add 25 ml. of concentrated hydrochloric acid, and cool. If a considerable amount of insoluble matter is present, filter or centrifuge and discard the pre-
cipitate. Saturate with sulfur dioxide, add 1 gram of hydroxylamine hydrochloride, warm on steam bath for 15 minutes, and set aside for 48 hours. Filter with suction through asbestos mats over glass won1 in 12-mm. filter tubes. (We bend a piece of 4-mm. tubing, insert one end through a rubber stopper in the filter tube, and rest the other end on the bottom of the Erlenmeyer flask. This eliminates the tedious, and sloppy, manual transfer to the filter tube.) Rinse the Erlenmeyer flask several times with water, allowing the washings to go through the filter. Wash the filter tube carefully inside and out. Mount filter tube in a suitable suction flask or side arm test tube. Rinse the flask carefully with 5 to 10 drops of bromine solution (1 ml. of bromine, 60 ml. of concentrated hydrobromic
acid, 40 mi. of water), transfer to filter tube, and fill tube with water. After a few minutes draw solution through the mat and rinse the flask and tube with several small portions of water, combining washings with the solution. Add a few drops of 5% phenol to decolorize the solution and determine selenite by measuring the color produced by reaction with diaminobenzidine according to the method of C k n g . This procedure will convert selenium in biological materials into selenite and 7. appears to be quantitative \vithin It is, of course, necessary t o be reasona-
bly careful and to determine a reagent blank. I n the Cheng procedure, the use of more concentrated formic acid, thereby reducing the volume, gives more consistent results. In any case, the volume must be kept constant. Diaminobenzidine, synthesized by the method of Hoste (Z), gives us more consistent results than the commercial material we purchased.
(2) Hoste, J., Anal. Chim. Acta 2, 402 (1948). (3) Hoste, J., Gillis, J., Ibid., 12, 158 (1955). ( 4 ) Olson, 0.E., personal communication, 1952.
LiTERATURE CITED
WORKmade possible by a grant made by the Tektronix Foundation, Portland, Ore., to the Chemistry Department of the University of Portland.
(1) Cheng, K., ANAL. CHEW 28, 1738 (1956).
O s 2
CARLW. BOKHORST JAhlES J. hfATTICE University of Portland Portland 3, Ore.
~~
Determination of Clo-Akylbenzenes
Determination of Isomeric Dimethyl Benzyl Alcohols
D. E. NICHOLSON and S. H. HASTINGS, Humble Oil 8, Reflning Co., Baytown, lex. CS- 105
D. E. NICHOLSON, Humble Oil & Refining Co., Baytown, l e x . Slit
I
No.
Slit Accuracy
Componenf I Range Name 1 Formulo 1 %
%
B.L.
AX or
Concn. glliter Length
Pts.
Av
mm
X or
(mm)
Y
Range
1
3,5-Dimethyl benzyl alcohol
CgH120
% % ~0-100 10.5 1 1 . 8 5 ~
2,4-Dimethyl benzyl alcohol
CgH120
0-100 f 0 . 5
2,5-Dimethyl benzyl alcohol
C~HIZO
-
3
Relotive Absorbancer-Analyficol ComponentlX
0.713 0.394
0.050 0.242 Material Purity:
P o i n t X
Calculofion:
Successive a p p r o x A
22.21~
18.49~
3
1,4-Dimethylbenzene
Base line-
Inverse m a t r i r G r a p h i c a l
Mafrix: 12.35~
0.020 0.202 0.048
0.017
0.345 0.882 0.252
0.202
3
0.998
Successive a p p r o x L
12.20p
0.690 0.109 0.219
0.088
P o i n t X
0.211 0.015 0.017
2 Material Purify:
0.139 0.294
Minimum of 99%
Instrumenf: Perkin-Elmer Model 11 2, NaCl prism Sample Phase: Solution in carbon disulfide
CS- 107
Range
-
Cell Windows: NaCl Absorbance Measurement:
Concn. gllifer Lengfh
Calculation:
mm
-
1 1 2 . 3 5 ~ 0.229 0 . 0 4 4 ~ 0.027
99.9%
Componenf
1,3-Dimethylbenzene
1 G.218 0 . 0 4 2 ~ 0.027
11.85p
0.068
Humble Oil & Reflnlng Co., Baytown, l e x .
2
1 0.202 0 . 0 3 9 ~ 0.027
1
0.110
S. H. HASTINGS and D. E. NICHOLSON
phenone
mm
Component/k
0.180
Determinatioir of 1r3- and 1 ,CDirnethylbenzenes Containing Benzophenone
-
0-100 k 0 . 5
Relative Absorbancet-Analyfical 21.32~
AA or Av
Concn. g/lifer Lengfh
~-
Cell Windows: NaCl Absorbance Measuremenf:
Mafrix:
20.16~
12.20p
(mm)
Instrument: Perkin-Elmer Model 112, NaCl prism Sample Phase: Solution in carbon disulfide
Insfrumenf: Perkin-Elmer Model 11 2, CsBr prism Sample Phose: Solution in carbon tetrachloride
Inverse m a t r i x Graphical-
or v 6.1. Pfs.
Formulo
-
Calculation:
X
Name
2
Base line-
Accuracy
No.
-
Cell Windows: CsBr Absorbonce Measurement:
CS-106
-I
f0.5 CsHlo
0-100
f0.5
i x K o-100 rto.5
5.95p
1 0.200 1 0 . 0 3 5 ~ 1 .oo
I
1 3 . 0 2 ~ 0.690 0.061~
1 1.00 1 1 .oo
Base line-
Inverse matrixGraphical-
P o i n t X Successive a p p r 0 x . X
Relofive AbsorbancerAnalyfical Matrix: ComponentlX
5.95p
13.02~
12.59~
1 2
0.219
0.007
0.002
0.000 0.000
0.272 0.002
0.002 0.277
3 Maferial Purity:
1.3- and 1,4-dimethylbenzenes, 99.5%; 98%
benzophenone,
VOL 31, NO. 12, DECEMBER 1959
2107
