the benzene of 99.93 mole % purity that was used for calibration. “he probability of any appreciable concentration of impurities being obscured by the benzene peak is, therefore, slight. The chromatograms of concentrgted inipurities on the poly (propylene glycol) 2025 and the benzylbiphenyl (Figure 1) columns exhibited three and one peaks: respectively, arriving before peak 1. As the total concentrations of the component(s) producing these peaks are very probably appreciably less than 1 p.p.m., no attempt was made to collect and identify them. Most of the Zmethylhexane was found in peak 2, but a small amount was also found in peak 3. This is a direct consequence of the fact that the Zmethylhexane had not been completely eluted with the other components of the first peak, before the components of the second peak of the pair began to elute. Similarly, most of the n-heptane was found in peak 4. but a small amount was found in peak 5. However, as long as both peaks of each pair are trapped from the same sample introduction, no error is caused by this partial failure to resolve the components. Thii procedure was followed in each of these cases. In Table 11, therefore, the qualitative and quantitative analyses of the impuritiea have been tabulated on a cumulative basis, without regard to the distribution of a component between two peaks. The concentrations are expressed as volume, weight, and mole per cent, all based on the whole benzene sample. A total of 20 impurities was identified-8 alkanes, 8 naphthenes, 1 aromatic, and 1 heterocyclic compound with certainty, and 1 alkane and 1 naphthene with good probability. Neither olefins nor carbon disulfide was detected. as was expected from their very low concentrations. The 501 p.p.m. thiophene determined here
with the 444 p.p.m. found by the isatin method. The average 4.4% of the impurities unaccounted for agrees well with the a p proximately 4% unaccounted for in
agrees fairly well
pesk 7. For all the specifically identified components present in concentrations e x d i g 0.007 mole % (1% of the total impurity), the average repeatability of the determinations was =tt3.9y0;the worst repeatability was *9.9%. For all the specifically identified components present in concentrations lesa than 0.007 mole yo, the average repeatability was +8.370; the worst repeatability was f 24.2%. Over-all r e peatability, calculated from the total impurities found, was ~k2.77~.The accuracies of individual analyses cannot be estimated; in fact, the only indication of accuracy is the total average recovery, 95.6% of the total impurity measured by the cryoscopic method, which is usually highly accurate for a sample of this kind. The occurrence in coke-oven benzene of 2,2dimethylpentane, 2 , M i e t h y l pentane, Zmethylhexane, 3,Mimethylpentane, 2,3dimethylpentane, %methylhexane, %ethylpentme, l,lsCms-!Mimethylcyclopentane, l,ttum+dimethylcyclopentane, and 1,&-2dimethylcyclopentane, which had been identified tentatively by Anderson and his associates (6,6), has now been established definitely. l,trans-2, &Trimethylcyclopentane has been identified in coke-oven benzene for the iirst time. (The occurrence of another trimethylcyclopentane and an octane is highly probable.) Eleven new products of the high-temperature carbonization of bituminous coal have, therefore, been established with certainty. ACKNOWLEDGMENT
The authors gratefully acknowledge the assistance of J. E. Friedline, who
helped to record many of the mass spectra; of the Chemical Service Section, Coal, Coke, and Chemicals Division, Applied Research Laboratory, for the ASTM analyses; and of the U. S. Steel Fellowship a t the Mellon Institute for the preparation of the bepzylbiphenyls and for the development of the combustion method for total sulfur. UTERATURE CITED
(1) Am. Soc. Testing Materiels, Phil-
adelphia, Pa., “Fuels, Petroleum, Arqmabc Hydrocarboq, Engine An& freezes,” p. 1252, Designabon D 850-55
1955. (2) Ibtd., Designation D 1015-55, p. 465, 1955. (3) Zbid., Designation D 1016-55, p. 480, 1955. (4) Am. Soc. Testin Materials Corn-
mittee D-2 on Petr8eum Products and Lubricants, “Proposed Method for Hydrocarbon Types in Gasoline by Mass Spectrometry,” 1956. (5) Anderson, J. R., Engelder, C. J., Znd. Eng. Chem.37,541 (1945). (6) Anderson, J. R., Jones, A. S., Engelder, C. J., Zbid., 37, 1052 (1945). (7) Deaty, D. H., Whyman, B. H. F., ANAL.CEEM.29,320 (1957). (8) Diclrinson, J. D., Eaborn, C., Chem.
(w)
& Znd. 1956,959. (9) Goldschrmedt, G., Mondsh. Chem. 2,433 (1881). (10) Kimura, S., Yasui, H., J . Chem. Sm. Japan, Pure Chem. Sect. 75, 163 (1954). (11) Matthews, J. S., Coggeshall, N. D., ANAL.Cana. 31, 1124 (1959). (12) Matthews, J. S., Cogges@, N. D.,
“The Removal of Impunties From Organic Compounds by Progr&ve Freezing,” Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Pittsburgh, Pa., March 3-7,
1958. (13) Schwab, F. W., Wichers, E., J.
Research Natl. Bur. Standards 32, 253 (1944). (14) StinsendBrfer, H., OeZ u. KO& 38, 193 (1942).
RECEIVEDfor review July 24, 1959. Accepted June 24, 1960. Division of Gas and Fuel Chemistry, 136th Meeting, ACS, Atlantic City, N. J., September 1959.
Extraction and Flame Spectrophotometric Estimation of Thallium in Urine WILLIAM B. STAVINOHA and JOE B. NASH Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, rex.
,An assay of thallium in urine by flame spectrophotometry by use of an organic solvent extraction with 2octanone has been developed. After wet ashing of ihe urine sample, thallium is extracted with 2-octanone from a 1M hydrobromic acid solution. The 2-octanone is then washed with hydrobromic acid solution (1M) and
aspirated directly into the flame. Known amounts of thallium added to urine samples were recovered satisfactorily by this method.
I
N THE STUDY of thallotoxicosk,
it
became necessary to determine aceurately the thallium content of small amounts of urine. Of the general
separation techniques, extraction methods seemed to offer the most promise. The dithizone extraction method was useful but was relatively slow and involved numerous extractions (a, 3). Methods utilizing ether have been described (1, 6 ) which are useful for polarographic and colorimetric eatimation, but the use of a solvent exVOL 32, NO. 12, NOVEMBER 1960
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Table 1. Per Cent Recovery after One Extraction of 10 p g . of TI and 0.5 pc. TIa4 Added to 25 MI. of Urine
Urine Samples 2
1
3
Ttylliym remaining In aqueous 0 . 0 2 0.01 0 . 0 2 layer Thallium remaining in wash solution of dilute HBr 0 . 0 3 0 . 0 4 0.04 Thallium in 2octanone 89.40 100.90 101.00
ttaction which could be run directly in the flame spectrophotometer has not been described. We found that the use of 2-octanone as an extraction solvent results in a quantitative isolation of thallium, allows the extract t o be run directly in the flame, and also increases the spectral emission intensity. The sensitivity of the method is such that an emission sensitivity equal to 0.05 pg. of thallium per ml. of solvent per scale division on the Beckman DU spectrophotometer is obtainable ( 4 ) . APPARATUS
CALIBRATION CURVE
A Beckman Model DU flame spectroTable II. Specificity of Method for Thallium in Presence of Other Ions
Added to 2.5 MI. of Urine Containing 10 pg. of Thalliuma co +* Ca 16 6 mg. 16.6 mg. AI big M!l;* Na 208.0 mg. Sn Se + 2 1 . 0 mg. Fe f a Cr f3 Bi +a
Ga
In
cu
Cd Ni +* Hg +2
+2
Zn
Pb +Z
Ten mg. added unless otherwise noted.
Table 111. Relative Sensitivity of Flame Emission of Thallium in Water and 2-Octanone
Relative Sensitivity p:./Ml./Scale Division Water 2-Octanone
Slit Width 0.04
0.38
0.05
Table IV. Stability of 2-Octanone Solution of Thallium
photometer with a Model 4300 photomultiplier and an oxyacetylene burner attachment was used. Because of the toxicity of thallium, a vent was used to remove the fumes. ZOctanone, practical grade, obtained from Eastman Organic Chemicals, was used as received. The solvent was saturated with HBr by shaking with an equal volume of 1M hydrobromic acid. A standard solution of thallium, 1 ml. of which was equivalent to 5 pg. of thallium, was prepared by diluting to 1 liter 5 ml. of a stock solution containing 1.234 grams of thallium sulfate per liter. A wash solution of dilute HBr was made by the addition of 5 ml. of 48% hydrobromic acid to 50 ml. of water distilled in borosilicate glass. CALIBRATION O F FLAME SPECTROPHOTOMETER
The emission of the solvent blank was set at zero, with the shutter open,
hfean Std. dev.
Table VI.
14.8 15.2 15 4 15 1 1 0 2
Table V. Replicate Determination of Thallium Added to Urine Thallium (0.5 mg.) was added t o 1 liter of urine, and 25-ml. aliquots were taken Total pg. added = 12.5 Total pg. Recovered 12 512.5 12.3 11.8 12.3 13.0 13.5 12.3 12.0
5 10
11 0 11.3 12.5 12.5 12.8 12.5 12.3 12.5 12.8 ~
~~
11.8 15.0 15.2 15.0 10.2
Mean 12.4 Std. dev. 1 0 . 8
Estimation of 5-, lo-, and 20-pg. of Thallium Added to 25-MI. Samples of Urine
TI, rg. Recovered
Added 5 10 10 10
5 10.8 11 10
5 11 3 10 10
Mean Recovery, pg.
Std. Dev.
5
0
10.2 20.1
16%
ANALYTICAL CHEMISTRY
A standard working curve in the range from 0 to 5 pg. was prepared by transfer of 2, 5,g,and 10-ml. aliquots of the standard aqueous thallium solution to 250-ml. Erlenmeyer flasks and dilution with water to 10 ml. One milliliter of 48% hydrobromic acid and 0.05 ml. of bromine were added. The solution was heated to drive off the bromine and then cooled. Ten milliliters of Zoctanone were added, shaken for 1 minute, and transferred to a Wml. separatory, funnel. When the phases separated, the lower aqueous phase was drawn off. The organic layer was shaken with 10 ml. of wash solution of dilute HBr and again allowed to separate for about 5 minutes. The organic phase was then transferred to a cuvette and the emission intensity measured on the flame spectrophotometer. The cwrve was linear up to the highest roncentration used, 20 pg. per ml. of thallium. PROCEDURE
Samples contained 15 pg. of thallium and were measured against a freshly prepared standard and blank 0 Time, After 24 Sample pg. Hours, pg. 1 2 3
by adjusting the dark currcnt control. The sample containing 5 pg. per ml. of thallium was adjusted to 100% transinittanrr by varying the slit width. The slit width for the thallium standard containing 5 pg. per ml. was between 0.030 and 0.040 mm. The photomultiplier was set R t full srmitivity and at 3 on the zww suppression dial. The acetylene .pressure was adjusted to 2.5 p s i . and the oxygen pressure to 10 p.s.i. The thallium emission was measured at 377.6 mp. The instrument was calibrated with the aid of a Beckman 2260 mercury lamp.
k0.5 dzo. 1
A 25ml. aliquot of urine was transferred to a 250-ml. flask, and 20 ml. of concentrated nitric acid were added. A small funnel was placed over the top, and the solution was boiled gently on a hot plate to the point of dryness. Five milliliters of water, 0.5 ml. of hydrobromic acid, and 0.05 ml. of bromine were added. The solution was heated, and the same method was followed as for the preparation of samples for the calibration curve except that 5 ml. of k c t a n o n e and 5 ml. of wash solution of dilute HBr were used. The flame spectrophotometer was adjusted so that the standard containing 5 fig. thallium per ml. gave 100% transmittance and so that the solvent blank was zero; then the emission of the unknown was measured. EXPERIMENTAL RESULTS
The completeness of the extraction of thallium from urine was measured by the use of radioisotopic TlW4. Ten micrograms of thallium and 0.5 pc. of radioisotopic TP' were added to 25 ml. of urine, and the samples were then digested and handled according to the assay procedure previously outlined. The thallium content of the various phases was determined by counting 1- or Zml. aliquots in a Mark
13 Model 3 RCL Scalcr equipped with a Geiger-Muller tube. One extraction was sufficient to remove all but 0.01 to 0.02y0 of the radioisotopic T P 4 from the digested sample, and the wash solution of dilute HBr removed only 0.03 to 0.04% radioisotopic Tim' from the thallium in the a-octanone phase (Table I). Table I1 lists the elements which were tested for possible interference with the w a y . The amounts far exceed in magnitude the amounts of these elements which would be present in 25 ml. of urine under most circumstances. No interference was observed with any of 19 elements in 10-mg. amounts, or in the amounts indicated in Table 11, when they were added to 25 ml. of urine containing 10 ug. of thallium. The increase in the flame emission of thallium by use of Zoctanone w a s measured by the following procedure. The flame spectrophotometer was adjusted as described so that the standard containing 5 pg. thallium per ml. of 2-octanone gave 100% transmittance, and the blank of P-octanone was zero. Then by changing only the dark current adjustment, the amount of thallium in water required to attain 0 to 100% transmittance bet,ween water
and samples of thallium was ascertained. This was 38.4 pg. per ml. of thallium (Table 111). This is a sevenfold increase in sensitivity due to the use of 2octanone as thc solvent. To measure the stability of the solution of thallium in P-octanone, samples containing 15 pg. of thallium were estimated after extraction and washing and then estimated again after 24 hours agaiwt a freshly prepared standard and blank. As shown in Table IV, the solution of thallium in 2-octanone is stable for 24 hours. Table V lists the results of replicate estimations of 25ml. aliquots from 1 liter of urine to which 500 pg. of thallium were added, each sample containing 12.5 pg. of thallium. The recovery of thallium from the 18 estimations ranged from 10 to 13.5 pg. of thallium per sample with a mean of 12.4 pg. and a standard deviation of *0.6 pg. The results of the estimation of 5, IO-, and 20-pg. amounts of thallium added to 25ml. samples of urine are listed in Table VI. The results indicate a sufficient accuracy and precision to warrant the use of this method as a quantitative estimation of thallium in urine. The use of Zoctanone greatly sim-
plified the preparation of the samples for flame spectrophotometry. Although evaporation of 2-octanone from the prepared samples was not troublesome, the samples were kept covered with glass until they were aspirated into the flame. The stability of the sample allows a number of samples to be extracted and later estimated at one time. Because of the rapid, sensitive, estimation of thallium in small amounts of urine, this method has proved to be very useful in the diagnosis of thallotoxicosis in both human beings and animals. LITERATURE CITED
(1) Sandell, E. B., “Colorimetric Determination of Tracea of Metals,’’ 3rd ed., 828, Interscience, New York, 1959. ( 2 bill, C. W., Peterson, H. E., ANAL. CHEW21,1268 (1949). (3) Stavinoha, W. B., Nash, J. B., Emerson. G. A., Federation Proc. 18.448 (1959). ’ (4) Ibid., 19,391 (1960). (5) Woolley, J. F., AnaEyst 83,477 (1958).
RECEIVED for review January 15, 1960. Accepted June 13, 1960. Presented in part at the Federation of American Societies for Experimental Biology in Chicago, April 11-16, 1960.
A Rapid Spectrophotometric Assay of Alpha, BetaUnsaturated Acids and Beta-Hydroxy Acids RALPH A. SLEPECKY and JOHN H. LAW’ Departments o f Biological Sciences and Chemistry, Northwestern University, Evanston, Ill.
b A spectrophotometric method for the quantitative determination of CY,@unsaturated aliphatic acids has been developed. In addition, @-hydroxyaliphatic acids and poly-p-hydroxybutyric acid may be quantitatively converted to the corresponding a,& unsaturated acids and determined spectrophotometrically. The principle of this method lies in the fact that the ultraviolet absorption maximum of a,B-unsaturated acids undergoes a strong bothochromic shift when concentrated sulfuric acid is employed as a solvent. The resulting absorption maximum lies within the useful range of commercial spectrophotometers and is sufficiently intense to provide a sensitive method of determinotion.
Present address, James B. Conant, Laboratory of Chemistry, Harvard University, Cambridge 38, Mass.
F
et nl. (2) studied the effect of concentrated sulfuric acid on the ultraviolet absorption of carbonyl compounds and utilized the bathochromic shift of the absorption maximum which these compounds show in strong acid for the estimation of the base strength of the carbonyls. The protonated species formed in strong acids absorb light at a higher wave length than the hnprotonated species. Thus the absorption maximum of benzoic acid is shifted to 261 mp from 253 mp in 70% in 100% HzSO,. This phenomenon previously has not been exploited for the estimation of carbonyl compounds which absorb light below the useful range of ultraviolet spectrophotometers. The use of concentrated sulfuric acid as a solvent permits a rapid quantitative method for assaying crotonic acid and related n,&unsaturated acids. The observation of this property of crotonic wid resulted from an attempt to deLEXSER
velop an assay method for @-hydroxybutyric acid and bacterial poly-8hydroxybutyric acid, both of which were known to give rise to crotonic acid on dehydration or heating. It seemed feasible to conduct the dehydration reaction in concentrated sulfuric acid, and to measure the ultraviolet end absorption of the resulting solution. Exploratory experiments with solutions of crotonic acid in concentrated sulfuric acid revealed that the absorption maximum had shifted to 235 mp under these conditions, and that the absorptivity was sufficiently high to provide a sensitive determination. Furthermore, the dehydration reaction was found to be quantitative, thus providing a convenient assay for @-hydroxyacids and poly8-hydroxybutyric acid. Previous methods for the estimation of 8-hydroxybutyric acid (6, 9) involved an oxidation to acetoacetic acid and determination of the acetone formed VOL. 32, NO. 12, NOVEMBER 1960
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