V O L U M E 24, NO. 3, M A R C H 1 9 5 2
569
Table 11. Effect of Temperature of Color Development on Apparent Boron Content of Standard Solutions Boron Found, y/Ml.O
Boron Added, y/hfl.
85' F.
100' F.
1.50 3.00 4.50 6.00 7.50 3.00
the same manner as the sample solution but with the plant material omitted, the percentage trunsmittmce of the sample solutions waa determined. Recoveries were run with known amounts of boron added t o alsike clover. The results in Table IV show that the method is satisfactory for determination of microgram quantities in plant material. The method has been applied succesfifuIly to the estimation of boron in soil extracts and water.
Values calculated from calibration curve prepared a t 70" F. DISCUSSION
Table 111.
Effect of Fluoride on Apparent Boron Content of Standard Solutions
Fluoride Added,
Boron Found,
y
7
2.5 y boron added
7.5 y boron added
2 5 5.0 7.5 10.0
2.48 2.48 2.42 2 40
7.50 7.35 7.25 7.00
Table 1V.
Recovery of Boron Added to Alsike Clover
Boron .4dded,
0 20 '3 0 60
so
Boron Found,
Recovery of Added Boron,
Y
%
13.2 33.4 52.5 72.9 33 7
101.0 98.5 99.5 100.5
The calibration curve for the proposed method is shown in Figure 3, C. T h e photometric accuracy, evaluated from the slope of the curve, varies between 90 and 30% transmittance from 2.4 t o 0.6% relative error for a 0.270 absolute photometric error. Maximum accuracy occurs at a boron level of about 7 micrograms. i ~ good s in the range from 2 t o although the accuracy is e~sent~ially 8 micrograms. I n comparison wit'h the widely used method with 98% sulfuril, acid, the proposed method has t'he advantages of greater rangc: and greater sensitivity. I n addition the reagent is easier to prepare and dispense, and the color developed is less sensitive to small changes in acid concentration, LITERATURE CITED
(1) h m ~ c Offic. . Agr. Chcmists, "Official Methods of Analysis," 7 t h ed., p. 117, 1950. ( 2 ) Berger, K. C., and Truog, E., 1x0. EXQ.CHEM.,ANAL.ED., 11,
cium hydroxide solution and dried a t 105" C. After the volatile matter had been carefully driven off over a burner, samples were ashed for 1 hour a t 600' C. Experiments showed that 1 0 0 ~ o recovery of boron was obtained by this method. Exactly 15 ml. of 0.36 S sulfuric acid were added t o the ash and the resultant solution was filtered. One milliliter of the filtrate wm transferred to a comparator tube and an exact uantity (approximately 10 ml.) of quinalizarin reagent was a d d e l The tube was stoppered, and the contents were well mixed and allowed to stand for 24 hours a t room temperature. .kfter the colorimeter had been adjusted to 100% transmittance with a blank solution prepared in
540 (1939). (3)
IIaohle, W., Scott, E. IT.,and Treon, J., Am. J . HYQ., 29A, 1 3 S - 4 5 (1939).
14) Mitchell, R. L., Proc. Nutrition Soc., 1, 183 (1944). (5) Olson, L. C., and DeTurk, E. E., Soil Sci., 50, 257 (1940). (6) Saint-Rat, L. de, Compt. rend., 227, 150-2 (1948). (7) Smith, G. G., Analyst, 60,735 (1035).
RECEIVED for review July 11, 1351. Acaepted October 25, 1951. Presented to the Division of Agricultural Chemistry and Food Technology of the Chemical Institute of Canada a t the annual conference, Winnipeg, June 19.51.
Separation of Organic Insecticides from Plant and Animal Tissues LAWRENCE R. JONES AND JOHN A. RIDDICK, Commercial Solvents Corp., Terre Haute, Znd.
0""
of the most difficult analytical problems encountered in insecticide residue, penetration, and translocation studies on plants, and toxicity studies on animals, is the isolation of the insecticide from interfering biological substances. The methods for analyzing the insecticides per se generally are sufficiently sensitive and accurate for microgram quantities. There are specific methods for isolating microgram quantities of some insecticides from biological samples (&?'), (9, IO),but they usually are long and complicated. A general method that is simple and rapid for isolating insecticides from fats, waxes, and other biological substances should find wide application. The primary purpose of this study was the isolation of Dilan [an insecticide containing a mixture of 2-nitro-l,l-bis(p-chloropheny1)propane and 2-nitro-l,l-bis(p-chlorophenyl)butane ] from the several types of interfering biological materials. However, the method was extended to other insecticides to show its general applicability. The original method for Dilan (9),based on a simple n-hexane extraction, was modified to include an additional extraction step. It was found t h a t acetonitrile Selectively extracted insecticides and many other organic compounds from n+
hexane. Other solvent pairs should also be satisfactory. However, they should meet the following requirements: The insecticide ahould be soluble in both solvents, but have a solubility preference for one of them. The extracted biological material should have a solubility preference for the other solvent. The two solvents should be mutually insoluble and low boiling. DISTRIBUTION OF INSECTICIDES BETWEEN n-HEXANE AND ACETONITRILE
The distribution between the solventa was determined for the following insecticides:
++%%,(9).1,2,3,4,5,6-hexrtchlorocyclohexane
Dilan, standard, 99 Lindane, standard 99 (19). \ - ' - I .
Chlordan, purifled technical grade, John Powell, Inc., and Velsicol Corp. Parathion standard, 99.8%, American Cyanamid Co. Methoxychlor, technical grade, 90.0%, E. I. du Pont de Nemours & Co., I n c DDT, technical grade, 90.4% p,p' isomer, Eimer and Amend,
570
ANALYTICAL CHEMISTRY
Colorimetric methods of analysis n ere used whenever possible bec:iuse of the ease of adaptability and rapidity. No attempt 15 a1 made to evaluate the eevei a1 methods reported for the same insecticide. The distribution of the insecticides betn een ncetonitiile and he\:uie is summarized in Tahle I. REAGENTS
n-Hexane, Phillips Petroleum Co.. commercial grade, redisdilled. Acetoilitrile, Siacet Chemical Division, U. S. Vanadium Corp., conimercial grade, redistilled, and saturated with n-hexane. ]ilari was isolated from 300 nil. of n-hesane solution by the ~ ) i ~ ) r e d udescribed re below. Place 100 ml. of acetonitrile in the separatory funnel containing thv insecticide solution, shake thoroughly for a few minutes, and allow the phases to separate. Drain the lower acetonitrile phase into a 500-ml. round-bot'tomed flask. Extract the hexane with three additional 100-ml. portions of acetonitrile. Combine the four acetonitrile fractions and discard the hexane layer after the fourth extraction. Occasionally, additional purification of the isolated insecticide is required. This is done by combining the four vxtractions of acetonitrile in a clean separatory funnel, shaking with 100 ml. of fresh n-hexane, alloiving to settle, and separating. The hexane must then be re-extracted with two additional 25-ml. portions of fresh acetonitrile. Discard the hexane and combine the washings of acetonitrile with the other acetonitrile in the 500-mI. flask.
'J'able I.
Distribution of Insecticides between Acetonitrile and Hexane at Room Temperature ~
Insecticide Dilan Methoxychlor Parathion Lindane Chlordan DDT
Solvent Ratio-Volume Ratio_________ Volume Acetonitrile-Hexane_1:3 3:5 1: 1 3: 1 o+ Insecticide in Acetonitrile (A) and Hexane (H) 4 I1 A H A H A H 2 96 4 98 81 19 91 9 2 98 96 4 91 9 81 19 96 87 13 68 32 70 30 4 94 70 30 6 86 14 68 3 2 78 22 59 41 4 1 59 34 66 76 25 40 60 57 43 32 68
.ittach the flask of acetonitrile to a condenser and evaporate the solvent just to dryness. Avoid overheating the residue. Traces of acetonitrile do not interfere with the determination of nilan. Dissolve the residue in ethyl alcohol and transfer quantitatively to a 10-ml. volumetric flask. Ililute to volume with ethyl alcohol and mix. Determine nilan by the method of Jones and Riddick (9). EXTRACTION OF SPR4Y RESIDUE FROM PLAAT TISSUE
IVeigh a suitable amount of the plant tissue to contain, preferably! 1.0 to 6.0 mg. of Dilan and place in a widemouthed, screwcap jar. Add 300 ml. of n-hexane and strip the residue by shaking the sealed container vigorouslv for a feij- minutes. Filter the hexane into a 1-liter round-bottomed flask. Wash the tissue in a like manner with two additional 100-ml. portions of n-hexane, filtering into the flask as before. Concentrate the strippings on the still to 250 to 300 ml., allow to cool, transfer to a 500-ml. separatory funnel, and rinse the flask with two 25-ml. portions of n-hexane, adding the rinses to the separstory funnel. Proceed IJ ith the isolation procedure. EXTRACTION OF INSECTICIDES IN TRANSLOCATION AXD EPENETRATION STUDIES
Strip the Dilan spray residue from the plant sample as described above. Place the stripped sample in a Waring Blendor, add 100 ml. of n-hexane, and blend for several minutes. Transfer the hexane-sample mixture qnantitatively to a sintered-glass funnel and remove the liquid by filtration with gentle suction. Wash the blender and funnel with additional portions of hexane
This work was undertaken because the isolation of organic insecticides from plant and animal tissues is one of the most difficult analytical problems. By the method outlined, DDT, lindane, parathion, chlordan, methoxychlor, and Dilan have successfully been separated in milligram and microgram quantities from plant and animal tissue. Some interference was encountered with substances believed to be carotene and vitamin i,but no interference was encountered with other types of extracted biological materials such as fat or wax. The method recovered the six insecticides with an accuracy of *5C& or better. It is rapid and convenient to use, and should be adaptable to the recovery of a large variety of organic compounds from plant and animal tissues.
Transfer the liquid from the flask to a 1000-mI. separatory funnel quantitatively by rinsing with hexane. Draw off any water phase present and discard. Transfer t'he hexane to a flask and concentrate to 200 to 250 nil. Allow to cool and return the concentrated Dilan solution to the separation funnel, using small portions of hexane to assure quantitative transfer. Proceed m-ith the isolation procedure. EXTRACTION OF RESIDUE FROM ANIIIAL T I S S U E
Grind the &sue to be examined through the food chopper. IVeiyh 50.0 grams of the minced tissue into a mortar and grind thoroughly with 200 grams of anhydrous sodium sulfate until the mixture is dry and powdered. Transfer the mixture quanitatively to a 60 X 180 nun. paper thimble and extract the tissue for 4 hours in the Soxhlet apparatus, with 150 to 500 ml. of n-hexane. Transfer the n-hexane from the Soxhlet apparatus to a 500-ml. still flask, rinsing the apparatus viith small amounts of n-hexane, and concentrate to 300 ml. (150 ml. of hesane are needed for each 25 grams of tissue extracted to keep the extracted material in solution.) Transfer the n-hexane to a 500-mI. separatory funnel, rinsing the flask with two 25-1111. portions of n-hexane. Proceed with the isolation procedure. EXTRACTION OF RESIDUE FROM DAIRY PRODUCTS
Butter. Place 50 grams (more if necessary) of butter, 300 ml. of hexane, and 100 ml. of ethyl alcohol in a 1000-ml. separatory funnel. Shake thoroughly until the butter has dissolved. Transfer the solution to a sintered-glass funnel and remove the liquid from the precipitated protein. Rinse the separatory and sinteredglass funnels with additional hexane. Transfer the liquid from the va'cuum flask to a 1-liter separatory funnel, rinsing with small amounts of hexane. Add 100 ml. of distilled water, shake thoroughly, and allow the phases to separate. This step removes the alcohol from the n-hexane. Discard t>he lower water-alcohol phase. Proceed with the isolation procedure. Milk. Add 100 grams of milk, 100 ml. of ethvl alcohol, and 200 ml. of n-hexane to a 1000-nil. separatory funnel and shake thoroughly for a feTv minutes. Divide the emulsion thus formed into t n o equal portions and transfer to two 250-ml. centrifuge bottles. Centrifuge samples for 5 minutes a t 1000 to 2000 r.p.m. Without disturbing the lower aqueous phase, draw off the hexane phase into a 1000-ml. separatory funnel using mild suction. Add 100 ml. of fresh hexane to each of the centrifuge bottles, stopper, shake well, centrifuge, and remove as before, combining the hexane layer with the other in the separatory funnel. Repeat the hexane extraction a third time. Transfer the hevane extract to a distillation flask and concentrate to approximately 250 to 300 ml. Transfer the concentrated Dilan quantitatively to a 1000-ml. separatory funnel. Proceed with the isolation procedure. EXTRACTION AND ISOLATION OF OTHER INSECTICIDES
Methoxychlor, 1,1,1-trichloro-2,2- bis(p -methoxyphenyl)ethane. The distribution of methoxychlor between hexane and acetonitrile is the same as Dilan. The extraction technique used for Dilan was found to be applicable for methoxychlor. I t was noted that aliquots of acetonitrile containing the insecticide could be refluxed with methanolic sodium hydroxide in the dehalogenation step without evaporation of the acetonitrile prior to the addition of the alcohbl. No interference was encountered using the colorimetric procedure of Fairing and Warrington (Y).
571
V O L U M E 2 4 , NO. 3, M A R C H 1 9 5 2
cucuniixrs were untreated with any insecticide. They were Parathion, 0,O-diethyl-0,p-(nitropheny1)thiophosphate. Paraspecially selected for uniform size and shape, being approximately thion has Q favorable distribution between hexane and acetoni2 X 8inches. trile. The isolation procedure described for Dilan was used for Blanks Jvere run on the beans, apples, and cucumbers for each parathion, except that the original hexane extract was evaporated insecticide. There was an indication of a trace nniount of to a smaller volume initially in order to extract a larger percentage parathion on the beans. T o interference was ohscrveci from of insecticide by the acetonitrile. Acetonitrile does not interfere chlorophyll, wax, or any other biological materials. in the colorimetric method of Averell and Korris ( 2 ) . Parathion Kno\vn amounts of Dilan werr added to milk and l)uttcr, was determined directly in an aliquot of acetonitrile. Lindane, ~-l,2,3,4,5,6-hexachlorocyclohexane. Lindane has a tracted and analyzed (Table I Y ) . distribution ratio almost identical to that of parathion; the DISCUSSION same modification was used. Acetonitrile does not interfere in Extrnsive teats of this doutile estraction procedure lor plaiit the polarographic method of Fossuni and Riddick (8). Chlordan, 1,2,4,5,6,i,8,8-octachloro-2,3,3a,4,7,7a,-hexahydro- procedure for plant and animal tissues containing a known quantit>-of' Dilan have shown an average recovery of ahout N ~ , . 4,i-methanoindene. Chlordan has a n unfavorable distribution The following results are typical of those obtained for thc esbetween hexane and acetonitrile. However, the extraction traction and analysis of samples where a known amount (25 nig.) method was modified b y evaporation of the hexane volume to 100 of Dilan \vas added to animal tissue: 24.8,24.7,25.0,24.9.24.0, ml. and extracting with four 200-ml.portions of acetonitrile, so 25.0,24.8,24.8, 24.6, 25.0: mean = 24.85 or 99.40/0average that good recovery was obtained froni both plant and animal recovery; mean deviation = 0.015 mg. Similar precision 1v:is tissue. Acetonitrile does not interfere with the colorimetric proobtained with plant tissue wh(,n :I known amount of Dilan \viis of Ard ( 1 ) . Chlordan \vas det.ermined directly in an aliquot cedul,c% added. of the solvent. The use of n-hexane and acetonitrile for the isolation of Dilan DDT, l,l,l-trichloro-2,2-bis(p-chlorophenyl)ethane. DDT eliminates fats, waxes, and most other interfering biological subhas a distribution between the hexane and acetonitrile of the same order :is that of chlordan. The same extraction modification was stances. There are indications that small amounts of interfering substances are still not completely eliminated by this procedure, used as for chlordan. The acetonitrile solvent interferes with the particulai~ly those extracted from liver arid wilted or dry plant methods of Bailes and Pagne (S), Clayborn (41, and Stiff and tissues. These residual biological materials can be eliminated if Castillo ( 1 1 ) . It is necessary to evaporate the solvent completely the alcoholic sample, used in the color development, is divided before using any of these methods. into two parts of 5 nil. each; one is treated in the regular ni:innet~ APPLICATION ( 9 ) ,and the order of adding sodium hydroxide and ferric chloride reagents is reversed on the other. \Vhen this procedure is followed The method of extraction and isolation has been used for Dilan the color does not develop in the latter solution. This may then on hundreds of samples of a wide variety, both plant and aninial. he used as the blank and any foreign color present \vi11 tie present All fats and waxes and most pigments normally encountered ran in equal aniount,sin the blank and sample. be separated from the Dilan. The tests with parathion, chloldan, lindane, methoxychlor, The general applicability was tested by adding kno\vn amouiits and DDT were of an exploratory nature. of the insecticides to different animal tissues-i.e., liver, fat The method (8) for the determination of lindane was developed kidney, and muscle. The insecticides were estracted, isolated, for use in pharmaceutical preparations. It was used in this study and analyzed. The average results of the four analyses are given because it was the most convenient method available a t this tinie. in Table 11. I t is believed that a more favorable pair of solvents may he linon-n amounts of the insecticides were also added to gi'een found for chlordan ancl DDT. beans, cucumbers, and apples, extracted, and analyzed. The (ix-
~
average results of three analyses are given in Table 111. The beans and apples were purchased on the open market. The
Recover) from .inimal Tissue
Table 11.
(bo 0 grams sample melght)
Insecticide Dilan Methoxychlor Parathion Chlordan Lindane DDT
500.0y
10.0 10 0 1.0 10.0
Table 111.
Recover? from Plant Tissue
Insecticide Dilan Parathion Chlordan Lindane Methoxychlor
200 Oy 2 0 1 0
125 0
500 0-,
98.0
Recovery from Dairy Products
Butter Sample Wt. 50'Grams Dilan added, Recovery, Y
%
200 100 100
102
50
Recorery. ( 2 99 0 98 0 98 0
Amt. Added, M g . 1 0
Table IV.
50
Recovery, "/c 99.0 98.0 98.7 97.5 108.0 99.0
Amt. Added, M g . 10.0
100 98 110
90
Sample vol., mi. 200
100 100 100
200 200
Milk _ _ Dilan added, Recovery Y
70
200 200
100
100 100 100 50
99
95 100 103
90
ACKNOWLEDGMENT
The authors wish to thank Robert Rausch for many of the insecticide analyses, and John Fossum for the lindane analyses. They wish to acknowledge the cooperation of the John Po\\-ell Corp., Velsicol Corp., American Cyanamid Co., and E. I. clu Pont de Seniours & Co. in supplying the several insecticides. They also wish to thank the J. I\-. Davis Co., Terre Haute, Intl., for supplying the cucumbers. LITERATURE CITED
Aid, ,J. S., A l s . i I , . CHEM.,20, 858-9 (1947). Averell, P. R., and Xorris, 11.V., f h i d . , 20, 753-6 (19487. Bailes, E. L., and P a y n e , LI. B.. IXD.ESG. CHEM.,ANAL.ED., 17, 438-40 (1945). C l a y h o r n , H. V., .J. .i.ssoc. Ofiic. A4gr. Chemists, 29, 330 2 (1946). Clifford P. d., Ibid.. 30, 337 flS4T). Davidow. R.. f h i d . , 33, 131 (1950). Fairing. .J. D., a n d W a r r i n g t o n , H. P., Jr., Aduancrs C'hcm. Series, h-0. 1, 260-5 (1950). Fossum. J. H., a n d Riddick. ,J. A . , J . Am. Phann. dssoc., .Ski. Ed., 40,357 -8 (1951). Jones, L. R.. a n d R i d d i c k , J . .4.. -\X.I. S., Pogorelskin, 121. A , , and Haller, H. L.. Izu. ESG. CHEX, -%SAL. ED., 19, 51 (1947). Stiff, H. d.,a n d Castillo, J. C . , f b i d . , 17, 438-40 (1945). Toops, E. E., a n d R i d d i c k , J. A , , I L , CHEM., 23, 1108 (1951 j. RECEIVED August 1, 1951. Accepted August 20, 1951. Presented hefore Section 13, Pesticides, a t the X I I t h International Congress of Pure a n d Applied Chemistry, Xew Tork, N. Y., September 1951.
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