1850 test mixtures were selected because they are known constituents of coal tar and occur together in tar distillate fractions. Distributions of 53 transfers w-ere more than ample to resolve the mixtures into discrete bands, except for the toluidine mixture which required 90 transfers (Figure 2). The location of components in each distribution pattern could be judged from the previously known partition coefficients ( 7 , 8) of the individual components in the same solvent pair. I n the methylpyridine mixture, the position of the two constituents is readily observed because the 3-methyl isomer has no significant ultraviolet absorption a t 270 mp. The experimental recoveries for each mixture are summarized in Table I; the values represent the average of calculations based on the optical densities of three adjacent peak tubes. The recoveries are all within about 3 ~ 5 %of the known amounts; this is the usual accuracy of analyses by the countercurrent distribution method ( 1 , 5, I O ) .
a 0
Figure 3.
12
A N A L Y T I C A L CHEMISTRY
20 24 28 32 TUBE NUMBER
36
40
4 4 48
52
Separation of 2- and 8-4lethylquinoline
1. 2-Methylquinoline 2. 8-Methylquinoline Solid circle represents theoretical
(i) Golumbic, Calvin, and Goldbach, George, J . Am. Chem. Soc.. LITERATURE CITED
(1)
Baryy. C;. T., Sato, T.,and Craig. L. C . , J . Bid. Cherri., 188, 299 (1951).
( 2 ) Clark, W.hl., "Determination of Hydrogen Ions," p. 116, Baltimore, hId., Williams and Wlkins Co., 1927. ( 3 ) Craig, L. C., J . Biol. Chem., 155,519 (1944).
o.,
(4) Craig. L. C., and Post, H. i l h - . ~CHEM., ~. 21,500 (1949). (5) Golumbic, Calvin, Ibid., 22, 579 (1950). (6) Ibid., 23, 1210 (1951).
73,3966 (1951). ( S ) Golumbic. Calvin, and Orchin, Milton, Ibid., 72, 4145 (1950). (9) Golumbic, Calvin, Woolfolk, E. 0.. Friedel, R. A, and Orchin, Milton, Ibid., 72, 1939 (1950). (10) Yato, I-., Barry, G. T., and Craig. L. C., J . Bid. Chem., 170, 501 (1947). (11) Warshowsky, Benjamin, and Schanta. E. J., AXAL.CHEM.,20, 951 (1948). (12) Killiamson, Byron, and Craig, L. C., J . B i d . Chem., 168, 687 (1947).
R E C E I V Efor D review J u n e 11. 1952.
Accepted August 7 , 1452
Separation of Radioactive Iron from Biological Materials RALPH E. PETERSON A r m y Medical Seruice Graduate School, Walter Reed A r m y Medical Center, Washington 12, D . C. URIYG studies on radioactive iron metabolism, it was necess a r i to separate radioactive iron from large amounts of biological material. Kone of the published methods (2, 4, 5, 7 , 8, 10, 12) investigated gave quantitative separation of the iron from interfering salts prior to electroplating, when organic material containing a large concentration of calcium and phosphate was used. To circumvent these difficulties, advantage was taken of the Baudisch reaction ( 1 , 6) for the precipitation of iron with cupferron (N-nitroso-N-phenylhydroxylamine), chloroform extraction of the ferric cupferrate complex ( 3 , 9 , I I ) , and ashing of the cupferrate residue. This cupferrate separation eliminates the principal ions (phosphates, calcium) which interfere with the electroplating of iron in the alkaline medium produced during electrolysis. The procedure described outlines the cupferron method for precipitation and chloroform extraction of iron, and preliminary ashing of the tissues prior to separation of radioactive iron (Feb5or Fe59) from biological materials. METHOD
Preliminary Digestion of Biological Material. SERUM,RED CELLS,OR SMALLTISSUE SECTIOA-S.Place the serum, red cells, or tissue in a porcelain crucible or small evaporating dish, and cover with a few milliliters of concentrated nitric acid. SMALL ~ ~ N I M A L (MICE, S RATS). Place the animal in a large beaker, cover with a large volume of concentrated nitric acid (200 to 600 ml.), and allow to stand at room temperature 12 to 18 hours to effect solution of the animal. Concentrate the liquid digest to a convenient volume by heating on a hot plate, make up to a measured volume, and take an appropriate aliquot depending upon the anticipated radioactivity of the sample. Transfer this aliquot to a porcelain evaporating dish. FECES.Collect fecal specimens (100 to 200 grams wet weight) directly into Waring Blendor cups, add water, and thoroughly emulsify. Make this mixture to a measured volume in a glassstoppered graduate cylinder. Place an appropriate aliquot in a large beaker and cover with several milliliters of concentrated nitric acid. Single specimens may be conveniently emulsified separately, pooled (5- to 7-day collection) in a tared vessel, and
a weighed aliquot taken. These fecal aliquots in nitric acid are then evaporated to a small volume and transferred quantitatively to a porcelain evaporating dish. There are two circumstances that limit the size of the fecal aliquot that may be used: (1) excessive foaming on addition of nitric acid when too large a fecal sample is taken, and (2) carrier iron in excess of 10 to 15 mg. URINE. Evaporate one fourth or one half of a 24-hour urine to a small volume with several milliliters of concentrated nitric acid. Transfer quantitatively with repeated acid washes to a porcelain evaporating dish. To the porcelain dishes containing the nitric acid digests, add enough carrier iron solution (5.0 grams of ferric chloride dissolved in approximately 3 M hydrochloric acid and made to a volume of 100 ml.; 1 ml. contains 10 mg. of iron) to make a total of 5 to 10 mg. of iron. Evaporate the nitric acid digests in the porcelain dishes to dryness on a hot plate at a moderately low temperature at first to prevent spattering. Dry Ashing of Organic Material. Place the dishes containing the charred residue (no covers need be used) in a cool muffle furnace. Heat for 15 to 20 hours a t 500" to 600" C., with the temperature rising slowly. Remove the crucibles from the furnace, allow to cool, and if any trace of carbon still remains in the residue (the ash must be white), add a few milliliters of nitric acid, and again evaporate to dryness on a hot plate at a moderately low temperature a t first. If only a small amount of carbon is present the sample may be re-ashed on the hot plate a t a moderate temperature (200' to 300" C.). However, if a great excess of carbon remains it must be returned to the muffle furnace for several hours.
It is important to keep the temperature of the muffle furnace below 600' C. because ( a ) tissues containing a large amount of phosphates fuse a t high temperatures to a glassy mass which frequently entraps carbon that can then be burned off only with difficulty, and ( b ) a t excessively high temperatures the alkaline ash will attack the crucible to form insoluble silicates containing some of the iron, and these silicates cannot be dissolved in strong hydrochloric acid. \Then fairly large amounts of organic material are used, it is best to use porcelain crucibles rather than silica or Vycor be-
V O L U M E 24, NO. 1 1 , NOVEMBER 1 9 5 2
1851
cawe the porcelain is much more resistant t o attack by the alkaline ash. It is also important to use as little organic material ab possible, because when very large amounts of alkaline ash are present (20 to 40 grams) the porcelain dishes will be attacked.
Solution of the Dry Ash. After all of the organic material has been eliminated and only a n hite inorganic ash remains, add concentrated hydrochloric acid and heat the acid mixture j m t to dryness on a hot plate a t a temperature slightly below 100" C. (Small samples of serum, very small tissue sections, and red cells do not need to be carried through the following steps of the cupferron precipitation and extraction. These samples may now be carried through ironi 8tt.p 2, Preparation of Iron for Electroplating. ) Add 50 to 150 nil. of 6 Jf hydrochloric acid to the inorganic residue containing the ferric chloride, and heat on a hot plate a t a temDerature iust below 100" C. until most of the ash has been dissblved a n d the liquid has been concentrated to a volume of 20 t o 50 ml. The amount of 6 JI hydrochloric acid used and the final volume of the concentiate are dependent upon the amount of ash present.
Table I. Recoveries of Radioactive Iron Added to Hydrochloric Acid of F'arious Molar Strengths Hydrochloric Acid, hlolarity 0.3 0.6
1.2 2 : 3.0 4.0 6 0
Recovery Fesg, Counts/Minnte 1470 1500 1492 1478 1441 1140 900
Fe69 Recovered, % 97.5 100.0 99.6
98.0 96.0 76.0 60.0
Loss of E'ejg, % Filtrate 1.2 0.0 0.6 1.0 4.0 24.0 40 5
To this final 20- to 50-ml. volume of 6 J I hydrochloric acid, add water to a volume that will give a final concentration of 1.0 to 2.0 '$1hydrochloric acid. Heat a few minutes more if necessary t o dissolve any remaining inorganic material. Cool this solution to room temperature. The entire volume of solution may be used or an aliquot may be taken, depending upon the anticipated amount of radioactivity present. I t is necessary to heat the ash with the hydrochloric acid to convert all the iron to ferric chloride, and to change the insoluble iron phosphates formed during ashing to soluble phosphates. Precipitation and Extraction of Iron. To the cool (25' C. or below) 1.0 to 2.0 M hydrochloric acid solution containing the inorganic salts and iron, add with constant stirring a solution of 5% aqueous cupferron. Use approximately 1 ml. of cupferron solution for each 10 ml. of acid solution. Add an excess of cupferron-until a white precipitate forms on further addition of cupferron. Let this mixture stand at room temperature for 10 to 20 minutes and then transfer to a separatory funnel. ,4dd approximately 20 ml. of chloroform to the beaker in which the precipitation was carried out, rinse, and transfer to the separatory funnel. Shake vigorously for a few seconds, and then transfer the chloroform layer into a porcelain crucible (100-ml. capacity). Add another 15 to 20 ml. of chloroform to the hydrochloric acid solution and repeat the extraction. Add this chloroform to the first portion in the crucible. Evaporate the chloroform with a stream of air. Place the crucible in an oven or incubator maintained at a teniperature between 40' to 50" C., for 1 to 3 hours, to carry out the initial decomposition of the cupferrate. To the gummy residue in the bottom of the crucible add approximately 0.5 ml. of caprylic alcohol, place a cover on the crucible, and ash over a gas burner, high temperature hot plate, or in a muffle furnace. Raise the temperature gradually a t first to prevent any spattering of the cupferrate. The concentration of hydrochloric acid used for the precipitation is not critical but should not be greater than 2.0 M , because a t concentrations higher than thih) the ferric cupferrate complr.; begins to dissociate appreciably (Table I). The ferric cupferrate complex begins to decompose slow ly at temperatures much higher than 25" C., and for this reason, the precipitation should be carried out at a temperature below 25" C. With the chloroform extraction, only traces of phosphate or calcium salts are carried into the solvent. However, when large
amounts of biological material are used, it is advisable to remove traces of salts dissolved in the chloroform by washing the pooled chloroform extracts with a small volume of 1.0 to 2.0 JI hydrochloric acid before transferring the chloroform to the crucible preparatory to ashing. It is important to use a large crucible to ash the cupferrate residue and to add a few drops of caprylic alcohol before heating, because the cupferrate residue on dwompoaition vigorously effervesces. For the same reasons, it i p necessary to heat the residue carefully (40" to 50" C.) until gascous compounds cease to come off, and then to ignite it carefully :it a high temperature to the oxide of iron.
Preparation of Iron for Electroplating. To the ferric oxide residue in the crucible add a few milliliters of concentrated hydrochloric acid and heat on a hot plate at' a temperature below 100" C., just to dr ness. To the dried residue ad& 0.1 to 0.2 nil. of concentrated hydrochloric acid to redissolve the iron. To the ferric chloride solution in concentrated hydrochloric acid add approximately 10 ml. of a saturated aqueous ammonium oxalate solution, and mix. Transfer the oxalate solution to the electrolysis cell (no precipitate should develop). Rinse the crucible with one or two small washings of the saturated ammonium oxalate solution, and transfer this solution to the electroplating cell. The total volume of oxalate in the electroplating cell should not exceed 17 nil. with a cell of 25-m1. capacity.
It is necessary to add the 0.1 to 0.2 ml. of concentrated hydrochloric acid to the ferric oside residue to convert the iron to a soluble salt,, and also to keep the pH of the electroplating solution from rising too early during the electrolysis, with resultant precipitation of iron as the hydrate. Initially the pH of the electroplating solution is pH 4.0 to 4.5 and at the end of the plating period rises to pH 8.0 to 8.5. Preparation of Electroplating Equipment. Electroplating cells similar in design to those described by Vosburgh et a2. ( I d ) and Dunn ( 2 ) have been used. Copper planchats, 2.5-cm., have been used as the cathode, giving a surface area for electrodeposition of the iron of 2.0 sq. cm. S o significant selfabsorption of radiation from iron 59 occurs with planchets of this size if no more than 15 mg. of total iron are used. I t is necessary that the copper planchet's be very clean and smooth. These are prepared on the day used, by first washing with acetone, and then polishing to a smooth, shiny surface on a dental polishing wheel. The planchets are again washed with acetone and dried just before they are ready for use. Electroplating of the Iron.
Electroplate for 4 to 5 hours a t
1 i O to 200 ma. with a potential of 8 volts, and with the anode
(flat spiral of platinum wire) placed about 2.5 cm. from t,he copper cathode disk. I t is important to keep the anode-cathode distance constant, and to keep the surface area of the electroplated iron constant. The shorter the anode-cathode distance, and the smaller the surface area, the greater the counts per minute. The maximum deposition of iron will usually have been obtained after 3 hours. However, the osalate solution may be readily tested for iron before discontinuing the electroplating, by thc follon.ing procedure (using iron-free glassware): Remove approximately 0.5 ml. of the osalate solution and transfer to a test tube. Add 2 to 3 drops of thioglycolic acid, mix, and then add about 0.5 ml. of strong ammonium hydroxide. A pink color indicates an excessive amount of iron, and under such circumstances the solution should lie electroplated for a longer time. After the oxalate solution has been shown to give a negative test for iron, discard the oxalate and first lyash the copper planchet cont,aining the reduced iron with w-ater to remove all osalate, and then rinse with acetone. Then place these planchets in a desiccator until they are ready
1852
ANALYTICAL ash, recoveries average between 85 to 95%. samples the error of the method is 1 5 % .
Table 11. Recoveries of Radioactive Iron Added to Biological Tissues Wet Weight of Tissue, Grams 10 (rat) 50 (rat) 50 (rat) 75 (rat) 75 (rat) 100 (rat) 10 (feces) 75 feces) 6-Lour urine 24-hour urine 15 ml. of serum
Recovery of Fe69 Counts/Minute‘ 840 1450 766 860 3890 1280 1820 1650 1450 1800 1500
Feao Recovered, % ’ 97.0 98.5 95.5 98.0 99.5 91.0 100.3 93.5 96.5 87.0 100.0
CHEMISTRY With duplicate
LITERATURE CITED
Baudisch, O., and King, 1‘.L., J . I n d . Eng. Chem., 3 , 6 2 9 (1911). Dunn, R . W., J . Lab. Clin. Med., 37, 644-52 (1951). Furman, N . H., Mason, IT. B., and Pekola, J. S., ANAL.CHEX., 21,1325-30 (1949).
for counting. The radioactivity is measured with a thin mica end-window Geiger counter. RELIABILITY OF THE SEPARATION
Table I1 lists the recoveries obtained from various amounts of biological tissue with added radioactive iron 59. With serum, red blood cells, small rats, mice, and 10- to 20-gram feces samples, 95 to 100% recoveries are consistently obtained. However, with 12- to 24-hour urines that contain 15 to 40 grams of
Greenberg, G. R.. Humphreys, S.R., Ashenbrucker, H., Lauritsen, bl.,and Wintrobe, AT. AT., Blood, 2, 94-100 (1947). Hahn, P. F., IND.ENG.CHEV., - ~ A L ED., . 17,45-6 (1945). Lundell, G. E. F., and Hoffman, J. I., “Outlines of Methods of Chemical Analysis,” Kew S o r k , John W l e y & Sons, 1938. Moore, C. V., Dubach, R., LIinnich, V., and Roberts, H. K., J. Clin. Invest., 23, 755-67 (1944). Peacock, W. C., Evans, R. D., Irvine. I. \I-., Good, IT. XI.. Kip, -4. F., Weiss, S., and Gibson, J. G., Ibid., 25, 605-15 (1946). Rodden, C. J.. “Analytical Chemistry of the Manhattan Project,” New York, McGraw-Hill Book Co. 1950. Ross, J. F., and Chapin, M. d.,Rev. Sci. Instruments, 13, 7 - 9 (1942)
Sandell, E. B., and Cumniings, P. F., AN.AL CHEM.,21, 1356-8 (1949).
Vosburgh, G. J., Flexner. L. B.. and Cowie, D. B., J . B i d . C‘iienz., 175,391-404 (1948). RECEIVED for review May 7, 1952.
.iccevted August 18, 1952.
Determination of Sulfur in Petroleum Distillates by X=Ray Absorption A. Y. MOTTLAU
AND
C. E. DRIESENS, J R .
Standard Oil Development Co., Esso Laboratories, Linden,
K T H E past fex years, considerable work has been reported
I (2-5) on the adaptation of the x-ray Geiger counter spectrom-
eter to the determination of sulfur in petroleum distillates by x-ray absorption. While these procedures fail to match the accuracy of chemical methods, their timesaving features make them desirable for many applications. The Geiger counter spectrometer is not, however, ideally suited to x-ray absorption measurements. Because it is a single-beam instrument, unknown and standard must be compared in succession rather than simultaneously, thus introducing the possibility of error due to fluctuations in the beam intensity during the time required to make these absorption measurements. Furthermore, the geometry of the instrument is such that the beam must pass through the cell in a horizontal direction, necessitating a constant thickness of sample. Under these conditions, it is necessary to correct absorbance measurements for changes due to variations in sample density. This paper describes a procedure for the determination of sulfur in certain petroleum distillates using the General Electric x-ray photometer. Any improvement in speed, simplicity, and accuracy shown by this method over those cited is due entirely to instrumentation. The x-ray photometer was designed specifically for absorption work and, therefore, does not suffer from some of the limitations imposed by the Geiger counter spectrometer which was designed primarily for x-ray diffraction. The present method of analysis was named the “comparative method” by Zemany et al. (8), and their exploratory Rork demonstrated, as did that of S’ollmar et a2. ( 7 ) , the feasibility of applying it to the determination of sulfur in petroleum, as well as tetraethyllead in gasoline. Calingaert et al. (1) have applied the comparative method to the determination of tetraethyllead in gasoline, and the authors have profitably used their procedure, with modifications, for over a year. The method to be described has also been used for almost a year for the routine determination of sulfur in gas oils, heating oils, Diesel fuels, and lubricating oils containing no additives-any petroleum distillate whose normal sulfur content does not run much less than 0.1% and which does not contain any interfering substance.
.V. J .
EXPERIMENTAL
Apparatus. Absorption measurements were made with a General Electric x-ray photometer, Catalog No. 53283500-1, This instrument is described in detail by Rich and hlichel (6). Current was supplied by a 10-kv.-amp. Sorensen voltage regulator. A dummy load of 1200 watts in the form of tungsten lamps was connected to the regulator in parallel with the photometer. The sample, 85 grams, was held in the right-hand side of the double aluminum cell supplied with the instrument. (Each side of cell is 4.25 cm. square by 15.3 cm. deep.) A standard absorber consisting of a block of polystyrene, 3.5 cm. thick, was placed over the left-hand (reference beam) port of the photometer. A 50-mil aluminum block was placed over the right-hand (sample beam) port. A torsion balance (KO. 3015, The Torsion Balance Co., Clifton, N. J.), having 2-kg. capacity, and 0.1-gram sensitivity, was used to weigh the samples directly into the sample cell. Instrument Settings. Primary voltage 80. Emission current 10 ma. Amplification level 90. Calibration. Some of the samples used for calibration were prepared synthetically, while others were regular samples of known sulfur content. The sulfur contents of all the calibration samples were determined by a gravimetric procedure involving theuse of either the A.S.T.M. lamp (D 90-47T) or the Parr oxygen bomb (D 129-44). I t will be noted from the description given in Table I that these samples covered a wide range of carbonhydrogen ratios. The total x-ray absorption of each calibration sample was measured by the following procedure: The sample(85.0 i 0.1 grams) was weighed into the right-hand side of the sample cell. With the cell in position in the photometer, the null meter was brought to balance Kith the aluminum attenuator. The total absorbance of the sample was read in terms of the “drum reading,” which in turn signifies the mils of aluminum required from the attenuator to achieve balance. The drum reading is not the mils of aluminum equivalent to the sample in absorbance. The absorbance of the sample is actually equivalent t o the absorbance of the attenuator, plus the absorbance of the 3.5-cm. polystyrene block, minus the absorbance of the 50-mil aluminum block placed in the sample beam. Because, for this procedure the polystyrene block and the 50-mil aluminum block remain unchan ed, it is simpler and less confusing to think of them as part of t%e instrument, and to refer to absorbance simply in terms of drum reading. The difference between two drum readings can be correctly referred to as mils of aluminum.
