V O L U M E 25, NO. 11, N O V E M B E R 1 9 5 3 where d is the difference between duplicates and is taken for each of the n pairs. The duplicates referred to in Table I11 were in each case 5-ml. aliquots from the same final dilution of dextran solution, so that dilution errors are excluded from the calculation of the standard deviation.
Table 111. Precision of Anthrone Method
No. of pairs of duplicates Range of absorbance Range of difference between duplicates Standard deviation
Absorbance a t 6250 A. (1-Crn. Cell) Samples us. water Blanks us. water 97 345 0.001 t o 0 . 0 3 7 0.320 to0.888 (80% 0.500 t o 0.700)
0.000 t o 0 . 0 1 4 0,0024
0.000 t o 0 . 0 1 5 0.0029
As the standard deviation of the blanks (0.0024) is more than 80% of the standard deviation of the samples, the major source of error must be due to the relatively poor reproducibility of the blank. Although a value of the blank absorption is determined and subtracted from the absorptions of the samples, the variability of the blank remains in the corrected sample absorptions.
1661 The most likely esplanation of blank variability would seem to be contamination by minute quantities of extraneous cartanhydrate. LITERATURE CITED
(1) Black, H. C., Jr., ASAL. CHEM.,23, 1792 (1951). (2) Bloom, LV. L., and Willcox, R1. L. Proe. Soe. Exptl. B i d . Med., 76, 3 (1951). (3) Dreywood, R., IND. ENG.CHEM.,ASAL. E D . , 18, 499 (1946). (4) Gilman. H.. and Blatt, A. H., “Organic Syntheses.” Vol. 1. p. 60, New York, John Wiley & Son-s, 1941.(5) Greenewalt, C. H., IXD.ENG.CHEM.,17, 522 (1925). (6) Koehler, L. H., ANAL.CHEM.,24, 1576 (1952). (7) Loewus, F. A . , Ibid., 24, 219 (1952). (8) McCready, R. AT., Guggola, J., Silviera, T., and Owens, H. S.. Ibid., 22, 1156 (1950). (9) hIorri.3, D. L., Science, 107, 254 (1948). (10) Morse, E. E., ANAL.CHEM.,19, 1012 (1947). (11) Samsel. E. P.. and DeLaD. - . R. A.. Ibid. 23. 1795 (1951). . , (l2j Siefter,’ S., Dayton, S., Novic, B . , and ’Rfuntwyler, E., Arch. Biochem., 25, 191 (1950). (13) Viles, F. J., Jr., and Silverman, L., ASAI.. CHEX.,21, 950 (1949). RECEIVEDfor review March 2, 1953. .-lccepted .-lugust 18, 1953. Presented before the Division of Analytical Chemistry a t the 124th Meeting of t h e AMERICAN CHEMICAL SOCIETY, Chicago, Ill. T h e mention in this article of firm names or commercial products under a proprietary name or names of their manufacturer does not constitute a n endorsement of such firms or products b y the U. S. Department of Agrirulture.
Determination of the Gamma Isomer Content of Benzene Hexachloride By Chlorine-36 Isotope Dilution Method JOHN T. CRAIG AND P. F. TRYON’ Commercial Solvents Corp., Terre Haute, Ind. W. G. BROWN L‘niuersity of Chicago, Chicago, I l l . Considerable disagreement among a number of analytical methods for determination of the gamma isomer of benzene hexachloride led to the development of a new isotope dilution method using chlorine-36-labeled pure gamma isomer as a tracer. A known quantity of chemically pure radioactive gamma isomer of benzene hexachloride, labeled with chlorine-36, is added to a known weight of a technical benzene hexachloride sample of unknown gamma content. A sample of pure gamma isomer is then isolated from the mixture by an extraction
T
HE industry has been without an absolute analytical method for the determination of the gamma isomer content of technical grade benzene hexachloride. Infrared, polarographic, and chromatographic techniques have been used, but considerable disagreement has occurred a t times when different laboratories have attempted to check each other. It was the purpose of the work described herein to develop an isotope dilution method of analysis which is absolute and precise. A successful absolute method was developed which is practical and requires no special technique. Its standard deviation was found t o be 3 ~ 0 . 2 % in gamma isomer content. The time required for a single analysis is about 4 hours. Four determinations can be made simultaneously in an 8-hour day. 1
Deceased.
procedure. The specific act- :ity of the isolated material is compared to that of the original labeled isomer. The standard deviation of the method is 1 0 . 2 yoin gamma isomer content. Four determinations can be made by one analyst in an 8-hour day. This analytical procedure can be classified as an absolute method and is being used as a referee for other routine methods for the determination of the gamma isomer. It is being used to analyze benzene hexachloride samples having gamma contents ranging from 1 to 5096. Trenner e t al. ( 7 ) developed an isotope dilution method for the determination of the gamma isomer of benzene hexachloride using deuterium-labeled gamma isomer as a tracer. The method herein described is different in that a radioactive isomer has been used as the tracer. Throughout the experimental procedure, the chemically pure labeled gamma benzene hexachloride is referred to as the labeled standard. PREPARATION OF LABELED STANDARD
The initial step in an isotope dilution method of analysis is t o prepare a chemically pure radioactive compound which is identical in structure to the compound to be determined. In the method described it was necessary to prepare a quantity of pure
ANALYTICAL CHEMISTRY
1662 gamma isomer of benzene hexachloride labeled with chlorine-36. Radioactive Chlorination of Benzene. This procedure is based upon the known rapid establishment of exchange equilibrium between chlorine and chloride ion in aqueous solution. Inactive chlorine bubbled through radioactive hydrochloric acid becomes active by exchange, and a near-quantitative transfer of radiochlorine into the chlorine phase ie achieved. The fact that interchange occurs rapidly between a halogen and it.s corresponding halide ion was demonstrat,ed experimentally ( 2 , 3, 5, 6) several years ago. Halford (4)later explained this mechanism of interchange as a formation and decomposition of the trihalide ion and established a definite lower limit for the velocity of the inkrchange. The apparatus used for the chlorination is shown in Figure 1. Place 15 ml. of benzene in the benzene reaction tube (tube 2, Figure 1). Using a 4-ml. pipet to which is attached a hypodermic syringe, place 4 ml. of aqueous radioactive 0.2 hydrochloric acid containing 12 microcuries of chlorine-36 in the chlorine exchange tube (1, Figure 1). Passordinary chlorine from the cylinder (3, Figure 1)for 30niinUtes through the system u p t,o the tube containing the radioactive hydrochloric acid in order to displace all the air in the system. Then, open the st,opcock above the radioactive hydrochloric acid tube and bubble ordinary chlorine into the hydrochloric acid at, the rate of 0.1 gram per minute for 6 minutes. Through an exchange reaction, the radioactive ionic chlorine in the aqueous hydrochloric acid exchanges instantly and nearly quantitatively with ordinary chlorine, EO that molecular radioactive chlorine along with excess ordinary chlorine passes into the benzene renction tube and becomes dissolved in the benzene. After all the chlorine has passed into the benzene, place a 150watt lamp 3 inches from the center of the benzene and allow it to remain there until the yellow-green color disappears. The reaction is then complet,e. Introduce a stream of air through the bubbler in the benzene tube, apply heat to the coil, and distill the benzene. The air stream will flush t,he vapors through the condenser. About 1 gram of solid crude radioactive benzene hexachloride remains after removal of all the benzene. Dry the cake in a 75" vacuum oven to remove any remaining benzene. Extraction of Gamma Isomer. Extract the gamma isomer from the cake wit,h 2 nil. of n-hexane saturated n-ith nitromethane by heating t o the boiling point and stirring thoroughly. Decant the extract into a 50-nil. beaker. Repeat the extraction. Wash the spent, cake with 2 ml. of cold n-hexane and combine the wash with the t,wo extracts. Evaporate the solvent using a heat lamp placed about 8 inches above the beaker. ilfter the solvent is removed, the gamma oil remains. Final Preparation of Labeled Pure Gamma Isomer. Add 3.5 grams of pure unlabeled ganima isomer to the gamma oil obtained above. Dissolve the mixture in 16 ml. of 95% 3A ethyl alcohol by heating and stirrin all the material dissolves, cool to 20" C. with constant Allow the slurry to stand in the bath for 15 minutes stals appear. Filter the crystals and wash with ethj-I The melting point of the dried crystals is 112.0-112.8" C. Repeat the recrystallization a t least two more times to be assured of removing all minute traces of benzene hexachloride isomers other than gamma. D r y the final crystals for 3 hours a t 70" C. in a vacuum oven to remove all traces of solvent.. The labeled standard is noiv ready for use.
Figure 1.
Chlorination Apparatus
the vial to remain in the bath for 15 minutes and stir the mixture occasionally to dissolve all the gamma isomer. Not all of the crude sample will dissolve. Cool the mixture in the 20" C. water bath (3, Figure 2) for 30 minutes with occasional stirring to allow crystallization of isomers other than gamma. Leave the cap on during the crystallization. Tare a 15 X 50 mm. shell vial and place in a Niederl-Siederl sulfur filtration apparatus (2, Figure 2). Filter the supernatant liquid from the crystallization through a filter stick into the tmared vial. Wash the crystals by adding 0.3 ml. of perchloroethylene and cooling to 20' C. xhile stirring. Filter the wash into the vial containing the original filtrate. Place the vial containing the filtrate and wash in an evaporat.ion tube inserted in the top of t'he heat,ing bath and evaporate the solvent, using a stream of air directed a t the surface of the supernatant solution. Adjust t'he air stream so that splashing will not occur. Most of the perchloroethylene mill be evaporated in 30 minut,es. Weigh the residue which is the gamma oil. The usual yield of gamma oil at this point is 300 to 450 mg. Crystdlize t.he gamma oil by dissolving in 0.8 ml. per gram of a 1 t o 1 mixture of 1,i-dioxane and n-butyl alcohol while heating and stirring until a homogeneous solution is obtained. Cool at 20" (1. for 15 minut'es in the Iyater bath, then scratch the walls of the vial to induce crystallization. .4fter the crystals appear, allow the slurry to stand in the bath for 10 more minutes, then filter in the Niederl-Niederl filtration apparatus. Wash the crystals with about 5 drops of cooled n-butyl alcohol. The melting point, of the crystals ranges from 100' to 112' C. after drying for 30 minutes in a 75" C. vacuum oven. Recryst'allize the mat'erial from n-butyl alcohol, using approximately 4 ml. of solvent per gram of crystals, by dissolving, then cooling to 20" C. as before. Filter the crystals, wash with about
DETERMINATION OF GAhIM.4 ISOMER
The apparatus used in isolating the sample is shown in Figure 2. Weigh approximately 120 =k5 mg. of the labeled standard into a tared 15 X 50 mm. shell vial and add approximately 1000 f 5 mg. of the unknown technical grade benzene hexachloride. Both the labeled standard and the unknown must be weighed accurately to 0.1 mg. Add 1.2 ml. of perchloroethylene, place a cap (made from a 5ml. beaker cut in half) on t.he vial, and insert the vial into the well of a 115' C. perchloroethylene heating bath (1, Figure 2). Allow
Figure 2.
Sample Isolation Apparatus
V O L U M E 25, NO. 11, N O V E M B E R 1 9 5 3 3 drops of n-butyl alcohol, then dry for 30 minutes in a 75' C. vacuum oven. Repeat the recrystallization, using 3 ml. per gram of 95yG3A alcohol. Dry the crystals in a is0C. vacuum oven for 2 hours. A yield of about 50 mg. is usually obtained, melting a t 112.0112.8" C. If the melting point is in this range, the crystals are ready for counting. If not, the recrystallization must be repeated.
1663 Table 111.
% Gamma (Average) Method of Analysis Partition chromatography Infrared Polarography C1-36 method
(Six C1-36analyses of technical BHC) Sample No. % Gamma 14.1
13.8 13.9 13.7 Mean Range Standard deviation
Sample 1 13 4 13 2 13 6 12 9
X =
Table I. Precision
14
Determination of Gamma Isomer Content of .40AC Samples ( 1 )
2
13.d 13 9 0.4 * o 1.5
Sample 2 1 52 1 48 1 87 1 42
Sample 3 48 2 50 7 47 8 50 5
A ( B - C) C
where
X
le mg. of radioactive standard added to samp& specific activity of the standard, or counts per minute of standard/mg. of standard counted C = specific activity of the isolated sample, or counts per minute of isolated sample mg. of sample counted
A B
= nig. of gamma benzene hexachloride in earn = =
EXPERIMENTAL RESULTS
Table 11. Analyses of Synthetic Mixture ((21-36 analyeie of a synthetic technical grade BHC) Sample No. -4nalyst" Gamma F o u n d , % 13.9 14 0 13.9 13.9 13.9 14.1 .4verage value Range Standard deviation
14.0 0 2 r O 09
.4nalj:st 3 5 Analysts 1 and 2 were experienced in running the analysis. was not an analytical chemist a n d had never perforiiled such a n analysis or made any previous practire runs.
Table I shows the precision of the analysis. Six consecutive gamma determinations were made on a composite batch of technical grade benzene hesachloride which had been thoroughly blended. All analyses were run by the same analyst. Table I1 shon s the results of the analyses of a synthetic mixture of the alpha, beta, gamma, delta, and epsilon isomers, and heptachloride present in portions comparable to technical grade benzene hexachloride. The calculated gamma content w s 14.0%. Samples uere assayed by several methods in a cooperative arrangement by a number of benzene hekachloride suppliers. Average values by each method are shown in Table 111. Another AOAC sample of technical benzene hexachloride distributed for infrared analysis had an average gamma isomer content of 13.24%. Chlorine-36 results shoued 13.28%.
COUNTING PROCEDURE
DISCUSSION
Counting is done in solution. A thin-milledglass, liquid-jacketed counter tube (25 mg. per sq. cm., 10-ml. capacity) is used, shown in Figure 3. -1 background count should be taken before counting a sample, with the counter tube filled with the same solvent used to dissolve the sample. Thip value must be subtracted from the count of both the sample and standard.
The isolation of the sample in an isotope dilution method requires simple technique, as the isolation does not have to be quantitative. The only quantitative operations required are weighing the standard and unknown, weighing the final isolated sample, and counting. This analytical technique can be applied to benzene hexachloride samples having a mide range in gamma content. The weight of the unknown sample to be analyzed should be increased or decreased according to its estimated gamma content, so that the ratio of labeled gamma added to ordinary gamma in the Pample u ill approximate the ratio described in this procedure.
iiccurately weigh the isolated pure gamma sample (to the nearest 0.1 mg.) into a 12-ml. snap cap vial, add exactly 10 ml. of acetone, and shake unt'il all the sample is dissolved. Siphon the solution int,o the counter tube with a hypodermic syringe and count for about 15,000 total counts. After count,ing the sample, flush out the tube three t,imeewith fresh acetone each time, then siphon drJ-. Check background again for 10 minutes to make sure the tube is decontaminat.ed. In a like manner prepare a solution of 50 mg., accurately weighed, of the original standard and count to the same approsimate total count as the sample. Calculate the results using the formula given below. Calculations. 1-se the following e q u a t i o n f o r c a l c u l a t i n g the results:
ACKNOWLEDG\f E>T
The authors are deeply indebted to Cecil Carr, CommercialSolvents Corp., for his valuable aid in working out a semimicromethod for isolating the pure gamma isomer from crude benzene hexachloride. LITERATURE CITED (1) Bowen, C. V., J . Assoc. Oi9ic. A g r . Chemists, 33, 774 (1950). (2) Dodson, R. W., and Fowler, R. D., J . A m . Chem. SOC.,61, 1215 f l R2R) . (3) Grosse, A. V., and Agruss, AI. S., Ibid.,57, 591 (1935). (4) Halford, R . J., Ibid., 62, 3233 (1940). (5) Hull, D. E., Shiflett, C. H.. and Lind, S. C., Ibid.. 58, 535 (1936j. (6) Long, F. A., and Olson, A . R.. Ibid., 58, 2214 (1936). I . _ _ _
\TO SCALER
Figure 3. Counting Apparatus Model J. T., N. Wood Couuter Laboratory, 5491 Blackstone Ave., Chicago 15, Ill.
(7) Trenner, N. R., Walker, R. W., Arison, B., and Buks, H. P., d N . i L . C H E Y . . 21, 285 (1949).
RECEIVED for review February 27, 1953. Accepted August 17, 1953. Presented before the Division of Analytical Chemistry a t the 122nd Meeting of the .\MERICAS CHE\IICAL SocIErY, Atlantic City, N. J .
