Determination of Traces of Selenium in Organic Matter Combined Spectrophotometric and Isotope Dilution Method WILLIAM J. KELLEHER and MARVIN J. JOHNSON Department of Biochemistry, University of Wisconsin, Madison 6, Wis. A, method i s described for the determination of a few hundredths of a part per million of selenium in organic samples. The method consists of a micromodification of a spectrophotometric method employing 3,3'-diaminobenzidine as the selenium(1V) reagent and incorporates an isotope dilution technique to compensate for the unavoidable losses that occur during the course of the analysis.
R
have emphasized the growing importaece of sele nium in animal nutrition (9, 10). Of particular interest is the vitamin E like action of selenium, first observed in rats (18) and chicks (4, 11),and later reported for a variety of other animals. At the time of the discovery of this new biological role for selenium, analytical methods were available for determining selenium in natural materials down to the several parts per million level encountered in chronic toxicity studies (1). Neutron activation analysis (7) waa the only available means for determining selenium at these extremely low levels until very recently when W a t kinson (14) described a fluorimetric method by which as little as 0.02 pg. of selenium could be determined. His procedure for estimating the selenium was similar to that described by Cheng (8) except that measurement was made of the fluorescence, rather than light absorption, of the dipiaselenole formed by reaction of selenious acid with 3,3'diaminobenzidine. I n his procedure, however, continuation of the oxidation steps beyond a somewhat arbitrarily defined end point, was reported to r e sult in a loss of selenium and an increase in interfering fluorescence. The colorimetric method of Cheng (S),although highly specific, does not have the sensitivity required by most current biological studies. Kirk, Rosenfels, and Hanahan (6) have shown that the range of utility of colorimetric methods in general, can be considerably reduced by the application of ultramicrotechniques. In the present study some of the principles set forth by these authors were utilized in the construction of capillary absorption cells and the ECENT REVTEWS
procedure of Cheng was modified to increase substantially its sensitivity. Failure to obtain consistent or quantitative recoveries of selenium from samples of natural materials necessitated the adoption of an isotope dilution technique. APPARATUS
Polarimeter cells (0.5ml., 5 cm.) were tried for measurement of absorbance but gave poorly reproducible results and were difficult to fill. Capillary absorption cells having a capacity of about 0.4ml. and a light path of 5 cm. were, therefore, constructed. Each ccll was made from a piece of 7/&ch Teflon rod, 2l/2 inches in length. A '/ginch bore was drilled symmetrically about the longitudinal a s k The ends were drilled out and tapped with a S/*-inch tap to a depth (about inch) that redured the length of the '/rinch bore to 5 cm. Ports for filling and emptying the cell were provided by two '/rinch holes drilled radially from the external surface to the central bore. These were located about 1 mm. inside the ends of the '/,-inch bore. Commercial polarimeter tube cover glasses, 15.5 mm. in diameter, wcre used for end windows. These were held in place by aluminum screw ends threaded to match the tapped portion of the tube. The m e w ends were about '/a inch long and had a shoulder, 7/* inch in diameter, in addition to the threaded portion. A central bore, */le inch in diameter, was drillcd through the screw ends to allow passage of light through the assembled cell. A ring gasket cut from '/&ch rubber sheet was placed between the end window and the screw end. The assembled cell was filled by adding the solution to the lower port of the ccll held in an inclined position, and slowly returning the cell to a horizontal position as the filling is completed. The use of this procedure, as well as the proximity of the filling port to the end windows, prevents the entrapment of air bubbles in the central bore during filling. A Cnry recording quartz spectrophotometer, Model 11, was used for all of the determinations. The standard cell rompartment of this instrument readily accommodated the capillary absorption cells without modification. Since this instrument employs a double light beam, two rells were required. The diameters of the aluminum screw
ends were made identical to the diameters of the glass or quartz cells normally employcd with the instrument. The spacers, adjusted SO that the cells were centered in the cell holders, supported the cells by their aluminum screw ends in the same manner that standard cells were supported. Similarly constructed capillary absorption cells as well as cell holders for use with the Model B and DU Beckman spectrophotometers are available from the Microchemical Specialties Co., Berkeley, Calif. Reaction tubes, used for development of the color and for the toluene ex-traction of the colored dipiasclenole, were prepared by sealing one end of a piece of 8-mm. glass tubing, 110 111111. in length. A 10-cm. length of 5 m m . glass tubing, one cnd of which had been drawn out to a diameter of about 1 mm., was used with a rubber syringe to remove the upper toluene lager from the reaction tube and transfer this solution to the absorption rrll. A thin-window counter was used for all of the radioactivity determinations. The samples were counted after drying on 5 sq. cm. stainless fiteel planchets. A well-type scintillation counter is more convenient and efficient, but was not available at the time the work was done. The apparatus for wct oxidations and distillations consisted of a 300-ml. pear-shaped digestion flaak to which could be connected, for distillation, (by a 24/40 ground glass joint) to either a &inch air-cooled condenser or a &inch water-cooled condenser. The water-cooled condenser bore, at its lower end, had a 14/35 joint for connection of a pear-shaped 100-ml. receiving flask. This flask, when used as a distilling flask, was connected to a water-cooled 3-inch condenser. Prior to use, all of the glassware used in the assay was washed with hot detergent solution, riqsed with laboratory distilled water, then immersed in a hot mix%ure of concentrated nitric and sulfuric acid (3:l) for a few hours. After removal from this misture, the glassware was rinsed several times with glass-distilled water and finally dried in an oven. REAGENTS
Laboratory distilled water that had been redistilled in an all-gla~sapparatus was used to prepare all of the solutions used in the determination and wherever VOL 33, NO. 10, SEPTEMBER 1961
1429
the procedure called for addition of water to the sample. Radioactive selenious acid solution. Dilute a solution containing Se76 (as selenious acid) with water so that 1 ml. contains approximately 0.01 yg. of radioactive selenium whose specific activity is on the order of several thousand counts per minute per 0.01 pcg of selemum. Selenium-free concentrated sulfuric acid. Dilute 100 ml. of reagent grade concentrated sulfuric acid with an equal volume of water, add 20 ml. of 48% hydrobromic acid and heat strongly until dense white fumes appear above the surface of the sulfuric acid. Sodium dichromate solution. Dissolve 50 grams of sodium dichromate in water and dilute to 100 ml. Standard selenium solution, 1.0 pg. per ml. Dissolve 0.219 gram of sodium selenite in water and dilute to 100 ml. Dilute the resulting solution 1:1000 with water. 3,3'-Diaminobeneidine solution. Prepare freshly by dissolving 0.0125 gram of the tetrahydrochloride in water and dilute to 10.0 ml. PROCEDURE
Oxidation of Organic Matter. Place an amount of material (not to exceed 10 grams) containing not less than 0.1 pg. of selenium in the 300-ml. digestion flask. Add 1.0 ml. (or more for samples of high selenium content) of the radioactive selenious acid solution. Then add 100 ml. of concentrated nitric acid, 10 ml. of 60% perchloric acid, and 5 ml. of selenium-free concentrated sulfuric acid. Allow the mixture to stand a t room temperature with occasional shaking until the foaming ceases. Apply heat from a microburner very cautiously until the initial rapid oxidation begins. Remove the heat and allow the oxidation to continue. After this reaction has subsided, connect the U-shaped air-cooled condenser to the flmk and apply steady heat from a microburner in a manner that results in the very slow distillation of the nitric acid over a period of a t least 24 hours. Remove the heat when white fumes appear in the flask, allow the mixture to cool, and add 1.0 ml. of the sodium dichromate solution. Re-apply the heat so that the contents of the flask are maintained a t a very gentle boil and no material distills from the flask. Remove the heat when the contents of the flask become orange. If this does not occur within 30 minutes after the addition of the dichromate solution, remove the heat, add 5.0 ml. of concentrated nitric acid and resume the heating. Repeat this nitric acid treatment a t 30-minute intervals if necessary. When the oxidation is terminated (appearance of orange color), allow the contents of the flask to cool, add 10 ml. of water, and boil until white fumes appear. Remove the heat and cool the flask to room temperature or below. Separation of Selenium. Add 10 ml. of water to the digestion residue, cool to room temperature or below, 1430
ANALYTICAL CHEMISTRY
then add 15 ml. of 48% hydrobromic acid. Attach the 6-inch condenser to the digestion flask and to the 100ml. receiving flask through their standard tapered ground glass joints, Distill the contents of the digestion flask until white fumes first appear. During the distillation, keep the distillate cooled to 15' c. or below with cold running tap water or with an ice bath. After completion of the distiilation, disconnect the receiving flask from the condenser, attach it to the 3-inch condenser and distill its contents until 1 to 2 ml. remain in the flask. Collect the distillate in a 50-ml. beaker covered with a piece of filter paper having a hole in its center large enough to accommodate the drip tip extending from the condenser. Keep the beaker cooled in an ice bath during this distillation. Add 20 ml. of concentrated nitric acid to the beaker and allow the mixture to stand a t room temperature for about 30 minutes. Then pass a slow stream of air, filtered through a glass wool plug, through the reaction mixture until all of the excess bromine is removed and the solution becomes almost colorless. Boil the solution over a low flame until its volume is reduced to not less than 5 ml. Then place the beaker in a vacuum desiccator containing anhydrous magnesium perchlorate and sodium hydroxide pellets, evacuate, and allow it to remain a t room temperature until all of the liquid has disappeared from the beaker. Remove the beaker from the desiccator, add 1.0 ml. of concentrated ammonium hydroxide, and replace the beaker in a vacuum desiccator containing concentrated sulfuric acid and anhydrous magnesium perchlorate. Evacuate and allow it to stand a t room temperature until no liquid remains in the beaker. Remove the beaker from the desiccator and add 1.0 ml. of water. Rinse the sides of the beaker by drawing the liquid up into a clean I-ml. graduated pipet and allowing the liquid to drain down the inner walls of the vessel. Using the same pipet, transfer a 0.5-ml. aliquot of the solution to a reaction tube for colorimetric determination and a 0.3-ml. aliquot of a tared 5 s q . cm. stainless steel planchet for radioactivity determination. Colorimetric Determination. Add 0.1 ml. of 1.ON formic acid solution to the reaction tube containing 0.5 ml. of the recovered selenium solution or 0.5 ml. of a standard solution of sodium selenite. Then add 0.2 ml. of the diaminobenzidine solution, mix, and allow the mixture to stand a t room temperature for a t least 30 minutes. After this period, add in succession, 0.2 ml. of 0.75N ammonium hydroxide solution and 0.5 ml. of toluene. Cover the reaction tube with a small piece of thin polyethylene sheet, grasp between the thumb and forefinger, and shake vigorously. Centrifuge briefly, remove the clear toluene layer with a rubber syringe and glass tubing prepared for this purpose, and transfer it to a 5-cm. capillary absorption cell. Place the cell in the
-
sample compartment of the spectrophotometer containing a similar cell filled with toluene in the reference cell compartment. Measure the absorbance of the solution a t 420 mp. Radioactivity Determination. If a well-type scintillation counter is available, use it to count (as liquid samples) aliquots from the recovered selenium solution and from the radioactive selenious acid solution. If only a thin-window counter is available, count the samples after drying them, mixed with an equal volume of 2% gelatin in 0.1N NaOH, below 50 C. on flat planchets. Calculation. The selenium content of the sample, calculated from the absorbance, is corrected for the recovery obtained in the isolation procedure, as determined by comparison of the radioactivity added to the sample with that recovered from it. The result is also corrected for absorbance of reagent blanks and for the (usually very small) amount of selenium added as Se75. RESULTS
Table I shows the results obtained with standard solutions of sodium selenite. Beer's law is obeyed over the range of selenium concentrations examined. The use of standard solutions made with selenium dioxide prepared by oxidation of the element with concentrated nitric acid and purified by sublimation gave identical results. The use of tenfold concentrations of formic acid and ammonium hydroxide solution in the assay system resulted in approximately a 1% increase in all of the absorbances.
Table 1. Calibration Data for Colorimetric Selenium Determination Selenium, rg. Absorbance 0.2 0.3
0.227 0.338
0.4
0.445 0.681 0.916
0.6 0.8
Presented in Table I1 are the results of several analyses of samples containing organic matter. DISCUSSION
The usual method of separating selenium from organic samples (1) gave very low and erratic recoveries when tested a t low levels (0.1 p.p.m.) of selenium from organic samples. Smith (IS) has discussed the relative efficiencies of various oxidation mixtures containing perchloric acid. We found that dichromate, besides functioning as a catalyst (IS) was a very convenient indicator, the orange color of hexavalent chromium appearing a t completion of oxidation. Gorsuch (4)
~
has reported good recoveries of larger amounts (I p.p.m.) of selenium with perchlorate oxidizing reagents. In the procedure we finally adopted, overall recovery of selenium was usually 70 t o 7575, but separate investigations of the oxidation, distillation, and concentration operations with radioactive selenium showed that losses of only a few per cent resulted from the first two steps. Treatment of the hydrobromic acid distillate with nitric acid, followed by evaportion t o dryness, however, resulted in losses of from 20 to 30% of the added radioactivity. Almost all of this loss could be attributed to the evaporation to dryness since nearly quantitative recoveries were obtained when the volume of the solution was reduced to only 5 ml. Complete evaporation was necessary to remove nitric acid, which would oxidize diaminobenzidine. Electrolytic oxidation of the bromide ion to elemental bromine as well as oxidation with 30y0hydrogen peroxide gave even lower recoveries of selenium. Analysis of samples containing known amounts of selenium uncovered another complicating factor in the procedure. In preliminary studies, the nitric acidtreated distillate was routinely evaporated to dryness on a steam bath. The results of these analyses showed that the radioactivity recoveries were consistently higher than the colorimetric recoveries. This discrepancy could arise from a discrimination between the selenium present in the sample and the added radioactive selenium, or it could be due to conversion of part of the selenium in the final residue t o some form that was unreactive toward diaminobenzidine. To test the latter possibility, 1.0-ml. aliquots of a radioactive selenious acid solution containing 0.418 pg. of Se per milliliter were placed in beakers, to which were added 5 m l . portions of either concentrated nitric or hydrochloric acid. The solutions were evaporated to dryness on a steam bath, or they were boiled for a few minutes and then evaporated to dryness at room temperature in a vacuum desiccator over anhydrous magnesium perchlorate and sodium hydroxide. One milliliter of concentrated ammonium hydroxide was added to each of the dried residues, and the solutions were re-evaporated to dryness in the same manner as that initially employed. Each of the final dried residues was dissolved in 1.5 ml. of water, and 0.5-ml. aliquots were removed for colorimetric selenium determination and for radioactivity determination. Table I11 shows that drying on B steam bath resulted in the highest total losses of selenium as well as the greatest difference between the colorimetric and radioactivity recoveries. The consistently higher radioactivity
Table It.
Selenium Content of Various Samples of Organic Matter
Additions Sample, Grams Dried torula yeast, U.S.P.., 10 None None Dried torula yeast, U.S.P., 25 None None Dried torula yeast, U.S.P., 50 None Dried torula yeast, U.S.P., 10 None 25 mg. high-Se yeastd (4.5 pg. Se) 50 mg. high-Se yeast (9.0 pg. Se) Dried torula yeast, U.S.P., 10 None 25 mg. high-Se yeast (4.5 pg. Se) 50 mg. high-Se yeast (9.0 pg. Se) Dried torula yeast, U.S.P., 10 None None 0.05 rg. Se (as NanSeOa) Torula yeast, feed grade,. 10 None 0 . 2 pg. Se (as Na&JeO,) Dried torula yeast, U.S.P., 10 None 5 g. Brewer's yeast, Dried torula yeast, U.S.P., 5 Strain Gf None Brewers' yeast, strain G,f 10 None None 0 . 5 pg. Se (as NatSeOt) None Debittered brewers' yeast,o 2 None hTone 1 . 0 pg. Se (as Nad3eOJ) L(-)-Cystine," 1
Selenium Concn. of ReFound,a SeIenium,a covery, % pg. P.P.M. 0.122 0.0067 ... 0.102 0.0047 ... 0.252 0.0055 ... 0.274 0.0064 ... 0.480 0.0045 ... 0.114 ... ... 4.49 9.51 0 ,202 4 .49
... ... ... ... ...
97.4 103
...
95.3 101
9 .27 0,104 0.094 0.148 0.138 0.330 0.108
...
96.0
0.0076 0.0268 0.0068
96 ...
0.637 1.318 1 ,248 1.17 1.743 1.963 2.008 2.03 3.19
0.0597 0.123 0.116 0.110 0.167 0.955 0.975 2.00 3.09
... ...
... ... ...
97.1
... ...
...
102
...
... ...
109 Corrected for radioactive selenium added to sample, * Corrected for reagent blank and for radioactive selenium added to sample. c Control No. 58828, obtained from the Lake States Yeast Corp., Inc., Rhinelander, 5
Wis.
Torula yeast grown on a synthetic medium containing radioactive selenite (6). Control No. 72571, obtained from the Lake States Yeast Corp., Inc., Rhinelander, Wis. f Brewers' yeast, dried, strain G, biol. lab. No. OB 2229, obtained from AnheuserBusch, Inc., St. Louis, Mo. Obtained through courtesy of Lake States Yeast and Chemical Division of St. Regia Paper Go., Rhinelander, Wis. The selenium content of this yeast, as determined by activation analysis, a t the Oak Ridge National Laboratory, was 1.02, 1.08, and 1.08 fig. per gram. * Lot No. 8134 E, obtained from General Biochemicals Inc., Laboratory Park, Chagrin Falls, Ohio. d
recoveries indicate that this procedure converted some of the selenite t o a form that did not react with the diaminobenzidine reagent. Smaller losses and better agreement between the two recoveries were obtained when the solutions were brought to dryness at room temperature in a vacuum desiccator. In the latter case, the colorimetric recoveries were slightly higher than the radioactivity recoveries. This discrepancy could very well have arisen from the introduction of extremely small amounts of selenium into the system either adventitiously or from the reagents. Boiling with concentrated hydrochloric acid is known to reduce selenate, a form of selenium unreactive toward diaminobenzidine, t o selenite. The results presented in Table I11 indicate that little, if any, selenate was present in the radioactive selenious acid solu-
Table 111. Comparison of Colorimetric and Radioactivity Recoveries Obtained on Evaporation of Solutions Containing to Dryness
Drying Procedure Steam bath ("0s solution) Vacuum desiccator ("01 solution) Vacuum desiccator (HCl solution)
Recovery, % Colon- Radiometric activity 31.8 44.7 43.8 67.5 78.2 81 .8 83.2
40.4 54.0 47.9 67.5 73.1 76.6 76.8
tion used in the experiments since only a very small increase in the ratio of colorimetric to radioactivity recovery was obtained following this treatment. VOL. 33, NO. 10, SEPTEMBER 1961
1431
The douhle distillation procedure was found to be the simplest method of eliminating sulfuric acid from the hydrobromic acid distillate. Since the sulfuric acid was carried into the distillate as a fincly dispersed aerosol, various types of fractionating heads were either ineffective in its removal or they resulted in a loss of selenium because of their relatively large holdup volume. Complete elimination of sulfate was considered preferable to using the precautions necessary (3) when sulfate is present. Termination of the oxidation mas clearly defined by the appearance of the bright orange of the dichromate ion. The addition of water to the oxidation residue and reheating to white fumes served as an effective means of rinsing adhering particles and droplets from the sides of the flask and ascending portion of the condenser back into the flask. The prescribed minimum 24-hour heating period in the presence of nitric acid is somewhat arbitrary. For 10gram samples of yeast, the oxidation could usually be terminated 30 to 60 minutes after the addition of sodium dichromate if the mixture had been previously boiled for 24 hours. Shorter heating periods with nitric acid made it necessary to increase the subsequent heating period to 1 to 2 hours. Since
the higher temperatures employed in this latter period increased the possibility of loss of selenium, preference was given to the procedure that reduced its duration to a minimum value. The absence of erratic readings in the results presented in Table I shows that no difficulty was encountered in the positioning and alignment of the capillary absorption cells. The results of the analyses reported in Table I1 show that this method is satisfactory for determining selenium down to lrvels of a few hundredths of a part per million. A number of samples were prepared by adding selenium, in amounts ranging from 0.05 to 0.09 pg., to 10-gram portions of dried torula yeast, U.S.P. These additions of selenium, both as inorganic sodium selenite and as selenium-containing yeast, brought the levels of selenium in the samples (including the selenium contained in the reagents) up to a p proximately 0.015 to 1.0 p.p.m. The recoveries obtained on the added selenium were between 95.3 and 103’%. The selenium content of dried torula yeast, U.S.P., and of feed grade torula yeast was too low to be determined accurately. Approximately one half of the total selenium found in these samples arose from the reagents and
the errors were such as to allow the results to be expressed only as orders of magnitude. REFERENCES
(1) Assoc. Offic. Agr. Chemists, Washing-
ton, D. C., “hlethoch of Analysis,” 6th ed., p. 473, 1945. (2) Cheng, K. L., ANAL.CHEM.28, 1738
( 1956). (3) Danzuka, T., Ueno, K., Ibid., 30, 1370 (1958). (4) Gorsuch, T. T., Analyst 84,135 (1959). (5) Kelleher, W. J., Gitler, C., Sunde, M. L., Johnson, M. J., Bsumann, C. A., J . Nutrilion 67, 433 (1950).
(6) Kirk, P. L., Rosenfels, R. S., Hanahan, D. J., aid.,19 355 (1947). (7) Leddicotte, b. W., Reynolds, S. A., Nucleonics 8, 62 (1051). (8) Patterson, E. L., hlilstrey, R., Stokstad, E. L. Ii., Proc. SOC.Expfl. Biol. M e d . 95, 617 (1857). (9) Schultze, M.O., Ann. Rev. Biochem. 29, 391 (1960). (10) Schwarz, K., Nutrition Revs. 18, 193 (1960). (11) Schwarz, K., Bieri, J. G., Briggs, G. M., Scott, bZ. L., Proc. SOC.Exptl. Biol. Med. 95, 621 (1957). (12) Schwarz, K., Foltz, C. hl., J . A m . Chem. SOC.79, 3292 (1957). (13) Smith, G. F., Anal. Chim. Acta 17, 175 ( 1957). (14) Watkinson, J. H., ANAL. CHEM.32, 981 (1960).
RECEIVEDfor review March 20, 1961. Accepted May 11, 1961. Published with the approval of the Director of the Wit+ consin Agricultural Experiment Station.
Determination of Sulfide in Cyanide by the Lead Sulfide Turbidimetric and Cadmium Sulfide Iod o met ric Methods K. K. GEORGIEFF Research laboratories, Shawinigan Chemicals, lid., Shawinigan Falls, Quebec, Canada
b Concentrations of sulfide in cyanide as low as 1 p.p.m. (calculated as anhydrous Na2S) can be determined turbidimetrically. The sulflde is precipitated with 125 to 300% of the theoretical amount of lead acetate, and the absorbance of the colloid determined with a spectrophotometer. The concentration of NazS is then read from a calibration curve. Total time required is 1 to 2 minutes. Methods for minimizing errors due to haze, colored impurities in the sample, instability of the PbS colloid, oxidation of Na2S, and variations in the concentration of NaCN are described. When concentrations of NazS are in excess of 0.01%, the sulfide may be precipitated as CdS and determined iodometrically after being washed free of cyanide.
1432
0
ANALYTICAL CHEMISTRY
C
ONCENTRATIONS of sulfide in cya-
nide greater than 0.01% can be determined with an error not esceeding 5 to 20% by precipitation of the sulfide as cadmium sulfide, followed by titration with iodine. However, some samples of commercial sodium cyanide contain only a few parts per million of sulfide, and the cadmium sulfide procedure is not applicable. For such low concentrations the lead sulfide turbidimetric method has been used ( I , 6). Visual comparison of the lead sulfide colloid with a series of known standards (1) introduces human error in judgment, especially when the solutions are colored. Furthermore, since the standards change after a few minutes, some error due to this factor can also be expected. In any case this is rather tedious and hence a rapid spectrophotometric method
seemed desirable However, before reliable and reproducible results could be obtained it was necessary to minimize errors due to haze caused by precipitation of insoluble lead salts, instability of the lead sulfide colloid, color of the sample, and variations in cyanide concentration. REAGENTS AND PROCEDURES
Lead Sulfide Turbidimetric Method.
1% filtered solution of reagent grade Pb(0Ac)Z. 3Hz0in water. 5% stock solution of NaCN similar in color to that to be analyzed and containing less than 1 p.p.m. of NazS calculated on 100% NaCN. 5% aqueous solution of Morningstar gum arabic, which has been filtered through Johns-Manville Celite No. 535 until haze-free.
