I n viw- of the fact, that the 2-naphtliylaniine cont'ent det'ermined for sample F did not meet the U. S.Food and Drug .4dministrat'ion specifications for certified Yellow AB (4, 7 ) , the analysis was checked using a modification of the mrtliod of Harrow (8). Duplicate determinations on 1-gram samples of dye gave 801 and 701 p.p.m. of 2-naphthylamine. These are in reasonable agrccment with the results ohtained by 0111' nletllod, The chromatographic system descrilled may find application in the purification and estimation of other amines. ACKNOWLEDGMENT r .
1I i r authors esprrss appreciation to
W. C. Hueper for many helpful suggestions offered during the course of this work. LITERATURE CITED
(1 ) Bonser, G. M . , Bradshaw, L., Clayson,
D. B.: "Carcinogenesis, Mechanisms of ilction," Ciba Foundation Symposium, Little, Brown, Boston, 1958. (2) Bonser G. M., Clayson, D. B., Jull, J. W., Lancet 261, 286 (1951). (3) Bratton, A. C., Marshall, E. K., J . B i d . Chem 128, 537 (1939). (4) Code of Federal Regulations, Title 21, Section 9, U. S. Government Printing Office, Kashington, D. C. (5) Deutsche Forsrhungsgemeinschaft, Rommission zur Bearbcitung des Lebensmittelfarbstoffproblems, Mitteilung 6, December 1955. (6) Evenson, 0. I,., Kime, J. il., Florest,
S. S., IND.EIUG.CIIEM., ANAL. ED. 9, 74 (1937). (7) Federal Register 24, 883 (1959). (8)Harrow, L. S., J . Assoc. O$c. Ayr. Chemists 34, 131 (1951). (9) Harrow, L. S., Jones, J. H., Zbid ., 37, 1012 (1954). (10) Howard, G. .4., Martin, J. P., Biochem. J. 46, 532 (1950). (11) Hueper, W. C., Acta U n i o n Internationale contre le Cancer 13, KO.2, 219 (1957). (12) Hueper, W.C., Wiley>F. H., Wolfe, H. D., J . Ind. H y g . Tosicol. 20, 46 (1938). (13) Wilheim, R., Ivy, A . C., Gastroenterology 23, 1 (1953). RECEIVED for review September 10, 1959. Accepted March 23, 1960. Division of Agricultural and Food Chemistry, 136th Meeting, ACP. At1:intic (:it!-, E. J., Septemb(3r 195Cl.
Determination of Oxygen-1 8 in Phosphate Ion MICHAEL ANBAR, MORDECHAI HALMANN, and BRIAN SILVER Isotope Deparfmenf, The Weizrnann Institute o f Science, Rehovoth, Israel
b A fast and accurate method for the determination of oxygen-1 8 in phosphate ion was developed io study the kinetics of the exchange reaction between inorganic phosphates and water. The method i s based on the separation of phosphate ion as trisilver phosphate, which is subsequently pyrolyzed a t 1000" C. to yield oxygen.
P
is precipitated b y the addition of silver perchlorate solution. Trisilver phosphate is soluble in dilute arid and silver oxide is precipitated by the addition of alkali to solutions containing silver ion. Therefore, the phoqphate solution is adjusted to p H 5.8 to 6.2 after precipitation, and the p H of the solution is not allowed to rise above pH 8 to 9 a t any time during the precipitation procedure. The method described here is less susceptible to errors of isotopic dilution than one involving equilibration of pota5sium dihydrogen phosphate with carbon dioxide (2, 3). The procedure involving pyrolysis of potassium dihydrogen phosphate in the presence of mercuric cyanide to yield carbon dioxide (5) failed t o give reproducible results when attempted. HOSPHATE ION
PROCEDURE
A solution containing 0.2 to 0.5 mmole of orthophosphate ion in approximately 5 ml. of water was treated with slightly
less than the theoretical amount of silver perchlorate solution. A drop of bromocresol green was added t o the solution and dilute (approximately 2.47 sodium hydroxide solution was added carefully until the end point 11-as reached (pH 3.5 to 5.4). Alternate d r o p of sodium hydroxide and silver perchlorate solutions were added, thus keeping the solution in the range of pH 3 t o 5 until precipitation was complete. The solution was finally adjusted to p H 5.8 to 6.2 with the use of indicator paper. This procedure avoided the addition of alkali t o a solution containing a large concentration of unprecipitated silver ions, which might form silver oxide because of a high local concentration of alkali. The precipitate mas centrifuged off, Ivashed with n a t e r three times, and dried in a vacuum oven a t 60" t o 70" C. Then it was transferred to a platinum crucible which was suspended by a platinum mire in a borosilicate glass vessel provided with a stopcock. The vessel was evacuated t o less than 0.5 micron and the crucible then heated for 10 minutes in a Mullard H. F. induction heating generator (Type KO. F 5/2) to 1000° C. (as determined by a Leeds & Northrup optical pyrometer). DISCUSSION AND RESULTS
The yield of oxygen was approximately 1 ml. at standard temperature and pressure from 100 mg. of trisilver phosphate. The yield (approximately 10% of the total oxygen present) was directly proportional to the weight of tri-
I.
Oxygen-1 8 Determination in Orthophosphates Atom yo Atom 7 0 0 ' 8 in Phosphate
Table
0 1 8 in Water 0.462
8.5
10 _. ~.
12 95 31 79
Direct pptn. 0.456 10 77 12.68
31 06
Through Ba3P0, 10 96 12 90 31 31
silver phosphate in the range 40 to 200 mg. and n as not increased by prolonging the heating for orer 10 minutes. The vessel was attached to a vacuum line and the oxygen transferred, by the use of a Toepler pump, to an ampoule ( 4 ) having a constriction and a break-off tip. The mercury in the pump v a s allowed to rise within 1 cm. of the contriction before the ampoule was se:rled. The oxygen n as determined directly in the mass spectrometer (Consolidated Engineering Coip., Model 21-401) by scanning masses 32 to 36. The crucible n a s cleaned after each determination by leaving i t in hot nitric acid (1 to 1) for about 5 minutes, washing in distilled water, drying, and heating in the induction furnace. To test the method, samples of phusphoric arid were prepared by hydrolyzing phosphorus trichloride in HzO1* (from the separation plant of this institute) and oxidizing the resulting phosphorus acid with elementary bromine. Silver perchlorate was added VOL. 32, NO. 7, JUNE 1960
0
841
directly to the reaction mixture, or the bromide ion was removed as follows: The solution n-as adjusted to p H 10 to 11 with sodium hydroxide solution and excess barium chloride solution added. The resulting precipitate of barium phosphate was centrifuged off, ivashed three times with water, and dissolved in the minimum amount of 2 N perchloric acid. The clear solution was run through a column of Dowex 50X4(H+) cation exchanger and the effluent treated with silver perchlorate solution as described above. The results of the phosphate and water (1) analyses are given in Table I.
The results following the barium phosphate procedure appear slightly better, although the number of samples analyzed is too small to allow significance to be attached to the results. However, in the presence of interfering ions, and in particular in the analysis of biological reaction mixtures, the barium phosphate procedure is advisable. The reproducibility of the method was checked by dividing a sample of trisilver phosphate into three portions which were decomposed and analyzed individually. Results were 0.412,0.414, and 0.415 atom % ' of oxygen-18.
LITERATURE CITED
(1) Anbar, M., Intern. J . Appl. Radiation and Isotopes 3, 131 (1958). (2) Bunton, C. A., Llewellyn, D. R.,
Oldham, K. G., Vernon, C. A., J . Chem. SOC.1958, 3574. (3) Cohn, M., J . Biol. Chem. 180, 771 (1949); 201,735 (1953). (4) Rittenberg, D., Ponticorvo, L., Intern. J . Appl. Radiation and Isotopes 1 , 208 (1956). (5) Williams, F. R., Hager, L. P., Science 128,1434 (1958). RECEIVED for review September 14, 1959. Accepted March 14, 1960. Investigations supported in part by a research grant (RG 5842) from the Division of Research Grants, U. S. Public Health Service.
Rapid Colorimetic Determination of 1 P. P.M. of Thiophene in Benzene Use of Permanent Color Standards H. A. BARNETT, C. E. BOLE, and C. F. GLICK Applied Research laboratory, U.
S. Sfeel Corp., Monroeville, Pa.
b The isatin method has been modified to permit rapid colorimetric determination of 1 p.p.m. of thiophene in benzene. A simple block comparator containing combinations of artificial color standards eliminates the need for a spectrophotometer. An unskilled operator can determine thiophene (down to 0.8 p.p.m.) in a sample of refined benzene in 10 minutes in the plant with very simple equipment. By comparison with a variation of ASTM D 1685-59T, the error of the modified procedure was -0.06 and - 0 . 0 9 p.p.m. of thiophene when the artificial color standards were 1 and 2 months old, respectively. The maximum statistical range of any set of determinations, by several operators, on a standard sample of benzene containing approximately 1 p.p.m. of thiophene was 0.04 p.p.m.; the average statistical range was 0.02 p.p.m.
C
benzene-toluene-xylene mixture from coal-tar light oil is usually washed Li-ith 66" Baume sulfuric acid and neutralized before distillation. The distilled benzene normally contains between 100 and 400 p.p.m. of thiophene. The thiophene content can be reduced to a very low level by rewashing with oleum(fuming sulfuric acid) and then with a strong caustic. A method
842
RUDE
0
ANALYTICAL CHEMISTRY
was required to control the washing with oleum prior to mashing with caustic to produce a product with a maximum thiophene concentration of 1 p.p.m. The most important level was from 0.8 to 1.1 p.p.m. of thiophene. The method should be as rapid as possible, yet yield results with a n accuracy within +0.05 p.p.m. of thiophene. The classical method for determining thiophene in benzene depends on reaction of the thiophene with isatin in the presence of concentrated sulfuric acid and an oxidizing agent to form indophenin, the concentration of which is measured colorimetrically (4). I n a standard version of this method (1) the color of the indophenin solution is compared with the colors from standard solutions of thiophene in thiophene-free benzene treated in the same way. These standards must be prepared aneiv for each test, because their colors are fugitive. The lowest concentration of a n y standard is 0.001 gram of thiophene per 100 ml. of benzene (approximately 11 p.p.m. of thiophene by weight). I n most of the modifications of the isatin method used industrially to measure less than 11 p.p.m. of thiophene in benzene the absorbance of the indophenin solution is measured with a spectrophotometer ( 2 ) . French described a method based on the use of a Lovibond Tintometer for the colorimetric determination of less than 25
p.p.m. of thiophene ( 3 ) . I n general, these methods are not convenient for plant use, because they are slow and require skilled operators and expensive equipment. With the method described, l p.p.m. of thiophene can be measured and these disadvantages are largely eliminated. REAGENTS
All chemicals were reagent grade unless otherwise specified. Thiophene-free benzene, prepared as described (8). Isatin Reagent, prepared as described (2). Allow the solution t o stand for a t least 72 hours before use. A small amount of isatin may subsequently crystallize from the solution, but quality is not impaired. After 2 months the reagent deteriorates and must be discaraed. Ferric Sulfate Reagent. Mull 0.020 gram of ferric sulfate, Fe2(S04h.zH2Or Gith approximately 15 ml.' of concentrated sulfuric acid (specific gravity 1.84) in an agate or mullite mortar until all traces of the sulfate have dissolved. (Use acid from a full bottle or one which has not been permitted to stand uncapped in moist air.) Transfer the solution to a 100-ml. volumetric flask, rinse the mortar with several small portions of concentrated sulfuric acid, add the rinsings t o the flask, and dilute to volume with acid. Store in a glassstoppered bottle and minimize contact with moist air.
