Isolation of Pentachloronaphthalene from Cottonseed Feed Pellets ROBERT T. BLICKENSTAFF and JOSEPH E. CALLEN M i a m i Valley Laboratories, Procter and Gamble Co., Cincinnati, O h i o
A method was needed for the isolation and positive identification of pentachloronaphthalene in cottonseed feed pellets. The isolation was carried out by ether extraction of the pellets, saponification of the ether extract, chromatography on alumina of the unsaponified portion, t w o liquid-liquid extractions of the chromatographic fraction containing chlorine, and crystallization from ethyl alcohol of the main fraction from the second liquid-liquid extraction. The material isolated has an analysis consistent with the formula C&,CI,, and its infrared and ultraviolet spectra, x-ray diffraction patterns, and melting behavior agree with those of authentic pentachloronaphthalene. By this method as little as 8 p.p.m. of pentachloronaphthalene in cottonseed feed pellets can be isolated and identified. A combination of the separation procedure through the stage of the chromatographic separation and an appropriate colorimetric determination should make a satisfactory quantitative method.
T
HE work described consists of the isolation of a trace ma-
terial from cottonseed feed pellets and its identification as pentachloronaphthalene. The pellet sample was suspected of having been contaminated with a pentachloronaphthalene-containing grease during the pelleting operation. Hyperkeratosis (X-disease) has been produced experimentally in cattle with pentachloronaphthalene ( 6 ) , a chlorinated naphthalene isolated from a German wood preservative ( ~ 5 )a~ lubricant additive described ae a highly chlorinated naphthalene (I), and a pelleted feed manufactured in a press lubricated by a grease containing chlorinated naphthalene ( 2 ) . Rather than follow the separation scheme of Hansel et al. ( 5 ) (two solvent extractions and three chromatographic separations), i t seemed advisable to saponify an ether extract of the pellets. then chromatograph only the unsaponified portion of the extract. The primary purpose of the saponification step was to reduce the amount to be chromatographed by removing the oil. (This oil is residual cottonseed oil in the pellets and amounts to 97 to 99% c of the ether extract of the pellets.) The material i s o l a t e d c h r o m a t o f 2.00 graphically was very impure, but 0 In after two liquid-liquid extractions and one recrystallization, pentachloroE 1.5naphthalene was obtained in a pure 4l 0 73 state. .L
. ( ) II
a EXPERIMENTAL
E x t r a c t i o n of P e l l e t s . Finely ground cottonseed pellets (21 pounds) were extracted with ethyl ether (3 gallons) for 8 hours in a stainless steel Soxhlet extractor. Without replenishing the solvent, three more 21-pound batches of the same sample were extracted similarly. The ether solution was evaporated and the last traces of ether were removed by passing nitrogen through the warm, liquid residue.
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W 0.5 ~
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Saponification of Ether Extract. The residue (1611 grams) from the ether extraction was heated under reflux for 2 hours in a solution of 575 grams of potassium hydroxide and 100 ml. of water in 7000 ml. of 3A alrohol (5% methyl, 95% ethyl). The hydrolyzate was diluted with an equal volume of water and extracted with petroleum ether four times. The combined petroleum ether solution was washed with water, then evaporated to constant weight. Chromatographic Separation of Unsaponified Extract. The unsaponified fraction (17.9 grams) was dissolved in 160 ml. of petroleum ether and chromatographed on an 8 X 115 rm. drypacked column of alumina (Fisher adsorption alumina, 80 to 200 mesh). This solution was followed on the column by 3500 ml. of petroleum ether. The eluate was collected in 500-ml. cuts, each evaporated separately and weighed. When cut 6 had been collected (see Figure 1 ) the column was further developed with 3 liters of 2 volume % ethyl ether in petroleum ether, 1.5 liters of 4 volume 70 ethyl ether in petroleum ether, 4 liters of 10 volume yo ethyl ether in petroleum ether, 3.5 liters of ethyl ether, 2 liters of 10 volume % 3A alcohol in ethyl ether, and 2 liters of 3A alcohol. Cuts continued to be 500 ml. in volume except for No. 25, which was 2 liters. Aspirator suction was applied a t the bottom of the column to maintain a reasonable flow of eluate, about 40 ml. per minute. (This undoubtedly caused some evaporation of the eluate. ) Descent of the desired fraction on the column was observed by means of its fluorescence under ultraviolet radiation. The Beilstein copper wire test indicated the presence of chlorine in cuts 18 through 28. The cuts were divided into fractions as shown in Figure 1 on the basis of weight distribution and appearance. JVeights of the two chlorine-containing fractions were 1.55 and 0.84 gram?, respectively. Liquid-Liquid Extraction of Chromatographic Fraction 4. Most of fraction 4 (1.43 grams) was separated in a 50-tube Craig separator ( 3 )using methanol and 2,2,4-trimethylpentane. Each solvent was freshly distilled, then saturated with the other; 10 ml. of each phase was used in each tube. The total contents of each tube were evaporated and the residue was weighed and checked for the presence of chlorine. The contents of tubes 24 through 38 (Figure 2, a ) were combined and subjected to a second extraction, identical to the first. The combined residues from tubes 23 through 36 (Figure 2,b) weighed 0.166 gram. Crystallization of Crude Product. The fraction obtained from
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V O L U M E 26, NO. 10, O C T O B E R 1 9 5 4 Table I.
Sample Cottonseed pellets Cottonseed meal Cottonseed meal plus grease
.
a
b c
Ether Extract, Grams 1611 1678 1427
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Separation of Cottonseed Samples
Unsaponi- ChromatoCrystalSecond L-L lired from graphic First L-L fied, Material, Fraction Extraction, Extraction, Ethyl 8100Grams 4, Grams Grams Grams hol, Grams 17.9 1.55 0.3147 0.1658 0.060 23.1 0.66 0,1007 0.0127 0 47.6 1.70 0.4633 0.2596 0.094 Calculated for pentachloronaphthalene
Analysis," %
H
C 39.83,39.79
1.27,1.18
40.oo,'40.12 39.98
0.9Q;i.22 1.01
CI 58.59,58.75 3.38b 58.68,58.46 59.02
llIicroanalyses by Galbraith Microanalytical Laboratories Knoxville Tenn. Analysis obtained o n sample from second liquid-liquid exkraction, as'no crystalline product was recovered from its ethanolic solution. Ethyl ether extract of meal; to this was added ethyl ether extract of 6 grams of a pentachloronaphthalene-containinggrease.
the Craig separator (0.166 gram) was dissolved in warm 3.4 alcohol, cooled in ice, and centrifuged. The crystals which collected were dried in varuo. i l s a ~ ~ s r sCalculated . for CloHsC1, 39.98% C, 1.01% H, 59.02y0 C1. Found 30.53, 30.i0YG C, 1.27, 1.18% H, 55.59, 58.75% C1. DISCUSSION OF METHOD
The chromatographic separation of alumina gave three major fractions and four minor fractions (Figure 1). An infrared spectrum of fraction 1 indicates that it is composed largely of aliphatic hydrocarbons. Fraction 1,the only major chlorine-containing fraction analyzed 9.99% chlorine, indicating a maximum of 1 i % pentachloronaphthalene, the remainder probably being hydrocarbon. Fraction 5 alpo contains chlorine, but its infrared spectrum indicates a much broader mixt,ure of compounds. Some 30 chromatograms \+-ererun on unsaponified fractions from similar batches of cottonseed pellets; all gave three major fract'ions corresponding to fractions 1, 4,and 7 in this separation, plus minor fractions in some cases.
WAVELENGTH. MICRONS
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Figure 3. Infrared Absorption Spectra
b. Second E x t r a c t i o n
T u b e Number
These spectra were obtained on 1% solutions of the samples in carbon disulfide, using a Perkin-Elmer hIodel21 recording infrared spectrophotometer equipped with a sodium chloride prism, and with carbon disulfide in the reference beam
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Figure 2. Liquid-Liquid Extractions
Fraction 4 and a commercial pentachloronaphthalene (Halowax 1014, Halowax Products Division, Union Carbide and Carbon Corp.) have distribution coefficients of 5.08 and 1.87, respectively, in 2,2,4-trimethylpentane-methanola t room temperature. This implies that a t least partial separation between the chlorine-containing and chlorine-free components of fraction 4 could be effected by a liquid-liquid extraction with these solvents. Using the 2,2,4-trimethylpentane-methanolsystem in a
50-tube Craig instrument (,3),the separation illustrated in Figure 2,a was obtained. The low values for tubes 32, 33, 36, and 35 causing sharp irregularities in the curve are unexplained. Emulsion difficulties, which sometimes have this effect, were not encountered. Tubes 24 through 42 contained chlorine, but only 24 through 38 were similar in appearance; the light yellow melt, from evaporation of the solvents, crystallized in large, radiating aggregates. Combined residues from tubes 24 through 38 were subjected to a second extraction identical to the first (Figure 2,b). Tubes 21 through 43 contained chlorine, but only residues from tubes 23 through 36 had the characteristic appearance; they were combined into a fraction which analyzed 39.41% chlorine. As verification of the prefience of still some hydrocarbon, a 1% solution of this fraction in carbon disulfide absorbed strongly in the in-
ANALYTICAL CHEMISTRY
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From w t t c From ootta From eomn Cylindrical I)( niekelLfiltered I
a.
b. c.
tallization in alcohol gave linc wlrit,e ~wedlcrof the chlorinated naphthalene in a pure state. In order to provide a blank and a known for comparison, two other samples were subjected to th! same separation. The first was 8; sample of cottonseed meal which had not been pelleted and, thFrefore, should contain neither grease nor pentachlorunaphthalene. The second i m s a sample of the same meal to which was added tt s m l l amount of the lubricating grease used in the pelleting operation. This grease is known to contain 3r commoreid mixture of chlorinated naphthalenes averaging about pentnehloronaphthalene. T s b k I contains the data on all three samples; the procedure used in the three separations nag the same throughout, except that in the case of the blank (cot,tonseed meal) no orystals were obtained from cold alcohol in the final step. As an additional kno\vn, comparison waR made also with t,he
