1976 Table I.
ANALYTICAL CHEMISTRY Comparison of Cleveland Open Cup Flash Point with Micro Flash Point
Material Diesel receiver C
Cleveland Open Cup Flash Point ( I ) , F. Micro Flash Point, F. Operator A Operator B Operator B Operator C ... 205 205 20.5
Spray oil A
305
210 300
Spray oil B
325
320
Spray oil C
340
Printing ink oil
360
345 340 360
410
430
435
SAE 30 compounded oil
Neutral oil
SAE 30 lubricating 011
Mineral oil
320
320 315 345 343
2in 313
340 340
360 360
...
420
...
435
410 44.3
445
450
465
...
475
470
4.55 4R0 460 470 470 450 49.5 480
SAE 20 lubricating Oil
205 305 305
500
450
4Ln
...
450
475 480
...
RESULTS
Results have been obtained on samples covering a wide range of flash points with good duplicability throughout. For the range from 200" to 500' F., which is the range of normal interest for flash points by the Cleveland open cup, comparative results on 10 samples are shoyn in Table I. A large number of various types of oils have also been tested in the course of regular analytical xork with good duplicability of results. COR-CLUSION S
Apparatus has been designed and a method developed for
obtaining reproducible flash points on a micro scale (0.3-ml. sample). I n the range 200" to 500" F., the values are essentially the same as those obtained using the Cleveland open cup procedure described in ASTM method D 92-46. The micromethod, as compared to the macro, requires the same amount of time and gives equal precision, but uses only 0.3 nil. of sample instead of 50 to 70 ml. The stated specifications must be closely followed, for the flash point values are very dependent on the dimensions of the apparatus. The design of the apparatus permits determinations in the open laboratory, because a dark hood is not required and the rewlts are not affected by normal air currents. ACKNOWLEDGMENT
The authors wish to express appreciation t o the Gnion Oil Co. of Califoriiia for permission to publish these data. LITERATURE CITED
(1) Am. SOC.Testing Materials, "Standards on Petroleum Products and Lubricants," Designation D 92-46, September 1951. (2) Ibid., Designation D 93-46. (3) Cannon, M. R., a n d Fenske, M. R., IND. ENQ.CHEM.,As.ir,. E D . , 10, 297 (1938). (4)Ettele, C., U. S.P a t e n t 1,554,993 (Sept. 29, 1925). ( 5 ) Levin, H a r r y , IND.ESG. CHEM.,ANAL.ED.,9, 147 (1937). (6) Levin, Harry, Morrison, A. B., a n d Reed, C. R., ASAL. CHEM., 22,188-91 (1950). ( 7 ) Ormandy, W. R., and Craven, E. C., J . Inst. Petroleum Tc~~hnol., 8, 145-80 (1922). ( 8 ) Ibid., 9 , 39-45 (1923). (9) Osmond, C. H., a n d Abrams, V. R., dtlantic Lubricator, 4, S a . 6 (February1921). RECEIVED for review June 17. 1552. Accepted August 18, 1552. Presented before the Division of Refining at the 17th Midyear Meeting of the American Petroleum Institute, San Francisco, Calif., May 12 to 13, 1552.
Colorimetric Estimation of Residual Benzene Hexachloride WENDELL F. PHILLIPS Beech-Nut Packing Co., Canajoharie, N . Y . The lack of a sufficiently specific and sensitive chemical method for the detection of microgram quantities of benzene hexachloride prompted the investigation which led to the development of this colorimetric method. When benzene hexachloride is refluxed with an excess of aniline, a mixture believed to consist of diphenylamine and dichlorodiphenylamines is formed. This mixture forms a violet color with an absorption maxima at 510 mp when oxidized with vanadium pentoxide in 5 0 q ~sulfuric acid. Beer's law is obeyed over the range of 2 to 120 micrograms of the gamma isomer. The method described is readily adapted to routine quality control analyses and should be useful to the food industry.
S
I S C E Slade (8) announced the insecticidal activity of the gamma isomer of benzene hexachloride ( Ij2,3,4,5,6-hexachlorocyclohexane) it has become one of the major chemicals used in modern agriculture. The analytical methods ( 1 ) for determining this compound are not adequate for residue analysis, except for the recently described Schechter-Hornetein ( 7 ) method. The method proposed belon- is a modification of the procedure prrsented by Fairing ( 3 ) before the Division of Agricultural and Food Chemistry, a t the 119th meeting of the ~ ~ M E R I C ACHEMICAL X SOCIETY.
Among the published data on the chemistry of benzene hexachloride is a study reported in 1887 by Neunier ( 6 ) ,in which he noted a reaction betn-een aniline and benzene hexachloride. Subsequent investigation revealed that, when a large quantity of gamma-benzene hexachloride is refluxed with aniline and the
reaction products are separated, as indicated in the procedure, a mixture of compounds is obtained which forms a violet color (absorption maximum a t 555 mF) on oxidation in 50y0 sulfuric arid (v./v.) containing 0.05 mg. per ml. of vanadium pentoxide. The mixture is believed to contain three components n-hich form color n-hen treated n-ith vanadic acid. One of them is a dichlorodiphenylamine and the other tvio have not been satisfactorily identified, but they may be diphenylamine and another dichlorodiphenylamine. One of the unidentified compounds produces a red color in the vanadic acid solution which has an absorption maximum at 510 mp. The dichlorodiphenylamine forms a purple color which has an absorption maximum at 555 mp. K i t h the same reagent, diphenylamine gives a blue ( olor which has an absorption maximum a t 595 mp. When microgram quantities of gamma-benzene hexavhloride
V O L U M E 24, NO. 12, D E C E M B E R 1 9 5 2
1977 (or 7 19/38) female ground-glass joint and stopper. Colorimeter. Glass beads are used as boiling aids throughout the procedure.
10
9
9
a
REAGENTS
si 7
Acid Celite. Adsorb on 100 grams of Celite 100 nil. of fuming sulfuric acid (15% SO3) containing 0.75 gram per kg. of vanadium pentoxide. Aniline. Redistill reagent grade aniline over a niixZ 6 ture of 10 grams of mossy zinc and 7 grams of magnesium turnings, for each 500-ml. charge of aniline. sp Store in a brown bottle with several lengths of 205 gage aluminum wire. Use an asbestos-wrapped, Drchiman DU Jpcct,fophlom+hr /.00.2 cm. E.// ground-glass jointed, Vigreux-type column for fractom. / O 7/m/. tionation, and discard the first sixth and last fourth Codr: -. 7 isomer of distillate. aniline purified in this manner will keep ---. a ,samer 4 for several months. 350 400 450 500 550 600 650 Celite (Johns-bIanville analytical grade or equivaMILLIMICRONS lent). Other inert filtering aids may be used, providing they do not adsorb any benzene hexachloride or Figure 1. .Absorption Spectra of Color Formed by Gamma and Alpha Isomers contaminate the sample. Ethyl Ether. ACS reagent grade; does not have to be anhydrous. It is advisable to check the ether are refluxed Tyith aniline, a higher percentage of the red colorbd'ore use. Mallinckrodt analytical reagent grade ether No. 0850 forming material is obtained and t,he color complex formed by these amounts of benzene hexachloride exhibits an absorption masima a t ca. 510 mM. The oxidation of diphenylamine to its blue quinoid imonium salt has been (6,9 ) it is \lc]iered that the colored body produced by the dichlorodiphen!-laiiiine is of similar StrUcture. The isomers of beiizenc hcsachloride when treated 10 under identical conditions do not yield the same intrnsitv of color. The. epsilon isomer produced 110%, 9 the alpha goy', thr delta 40%, the Iirts approu-
ha&bg::g$ ~
~ One volunle ~ of hydrochloric ~ acid, ~ c,p. (36 to 37% HC]) 3 volumes of distilled water. Pentane, Shell Co. I n order to avoid the difficulty of interference from impurities present in petroleum solvents, they were purified by through a 1.5 I2O The lower third of the column was packed n-ith a mixture of silica gel (28- to 100-mesh) and precipitated silicic acid (10 and 1 part, respect,ively) arid the remainder rras paclwd with 14- to 20-mesh silica
8
Table I.
Insecticides Producing Color
Compound Dieldrin Aldrin
7
hIicrograriisa
DDT
3000
6
zoo0
JnnO ..._
Aramltc 950 Toxaphene 825 Heptachlor 700 Gamma-chlordan 07.5 Technical chlordan 450 Technical BHC >8 Gamma-BHC 50 a hlicrograins of material necessary to form same intemity of color as produced by 30 micrograms of gainins-BHC.
5
w Z
U
4
a
I-
C-
mately 5%, and technical about 80% of the color produced by an equal amount of the gamma isomer. Other chlorinated hydrocarbons that contain alkali-labile chlorine may produce colors when refluxed with aniline and treated as indicated in the procedure. Table I shows the amounts of some of the other insecticides necessary to produce the same amount of color formed from 50 micrograms of gamma-benzene hexachloride. I n most cases the colors formed by other insecticides are characteristic of the blue color formed by diphenylamine and fade rapidly. Unlees exceedingly large amounts of towphene or chlordan are present, no interference from them is encountered.
I
4
a 3
ILL
bp
P
SPECIAL EQUIPMER-T
Air Condensers, 150-mm. length of 10-mni. glass tubing, fitted with a T 16/15 (or T 19/38) male ioint. Biichner Funnels, fritted-glass, coarse porosity, 20-nim. and 60-mm. diameter. Test Tubes, 16 x 150 mm., fitted with a T 16/15
1 1
PO
40
Figure 2.
60 80 MICROGRAMS BHC
100
Typical Standard Curves
1PO
140
f
ANALYTICAL CHEMISTRY
1978 gel. The life of the column was prolonged when Shell pentane was used instead of Fisher petroleum ether. Mallinckrodt and Baker's petroleum ether were not found satisfactory. Petroleum Ether, Fisher E-139, c.P.,boiling range 30' to 60 ". Silica Gel, 28-200 and 14-20 mesh, Sodium Sulfate, C.P. anhydrous, granular. The sodium sul-
Table 11. Product Peanut butter
fate was solvent-washed and dried before use to avoid possible contamination of the sample. Sulfuric Acid Solution, 5OyG,1 volume of sulfuric acid (specific gravitv 1.84) to 1volume of water. Sulfuric Acid Solution, 547, by volume. Sulfuric Acid-Vanadium Pentoxide, 0.05 mg. of vanadium pentoxide per ml. of 50% v./v. sulfuric acid solution. Vanadium Pentoxide, C.P. RecoFery of Benzene Hexachloride Added to Strippings anhydrous.
Aliquot, hI1.
BHC Added,
100 100 100 100 100 100 100 100 100 100
Sweet potatoes
50 50 50 100 100
(L) (T) (T)
100 100 100 100
(Lj
100
75 .5 0 50 50 50
100
50 50
100 100
dpplesauce
50
Sweet potatoes
(T) (T)
(T)
(TI
(L) (L)
(Tj (T)
(T)
(T)
(L) (T) (L) (T) (T)
(T) 50 (T) 100 (T) 100 (T)
50 50
50
50 50 75
100
(T)
--a
100 100
(L)
r-
Peas Bananas Vegetables and beef
(T) (T)
50 100
100 100 100 50 50 100 100
50 50 50
(L)
50
2
Apricots and apples
(Lj
(T) (T) (T)
100
ya
I IJ
(L)
Recovered,
Recovery, %
y
48 47 51 90 83 100 80 Q5 104 104
96 94 102 90 83 100 80 95 104 104
102 48 49 104 103 103 52.5 49 48 43 103 103 86.5 102
102 96 98 104 103 103 105 98 96 86 103 103 86.5 102
Average, '70 PROCEDURE
The procedure consists primarily of six steps:
94.4
101.5
98.5
46.5 47.5 95 i8
s9
48 49 104 102 103
96 98 104 102 103
102 100.5
102 100.5
101.5
L = Lindane. T = Technical BHC. Average deviation = 5.3%
a
Table 111. Recovery of Technical Benzene Hydrochloride Added to Products Indicated Product Peanu t butter
Sample Weight, Grams BHC Added,
y
Stripping ~ l i q u o t , BHC in T'olume, MI. XI1. Aliquot, y
100 100
100 100 200
350 350 330
Sweet potatoes
100
Applesauce
100 100 100 100
Apricots and apples
Vegetables and beef
100 100 100 100
100 100 100
100 100 100 100 100 150 150 300 300 100 100
250 250 230 250
250 250
100
2 50
100
250
Reco1.e~ y
%
164 164 1.55 150
46.8 40.0 50 50 40 40 40 47.6 47.6 47.2 47.2 88.7 86.7
36 27.6 36 33.5 34 25.4 29.5 26 32 36 29 71 63
93 93 97 97 77 77 100 68
37.2 37.2 38.8 38.8 38.5 38.5 40 27.25
96 96 84 84 50 50 69 69 60 60 72 72 57 d6 56 -ifi
38.4 38.4 33.6 33.6
27 72.5 26 5 71.2 29.5 76 34.5 88.8 26 67.5 31 80.5 92.5 37 24.5 90 42 109 49 127 38.5 113 37 111
20 20 27.6 27.6 36 36 86.5 86.5 22.8 22.4 22.4 22.4
16.5 19
32 34 45 39 86.5 80.5 23 23 23 23
76.8 69 72 67 85 63.5 73.8 54.5 67 76.8 62 80.2 73.6
82.5 95 116 123 125 108 100 92.4 100.8 102.8 102.8 102.8
.4verage,
61.8
79.7
113.6
103.6
102.2
1. Extraction of benzene hexachloride from plant material 2 . Separation of benzene hexachloride from other extractives 3. Reaction Jyith aniline 4. Separation of reaction products 5. Development of color 6. Measurement of the intensity of color produced The techniques described in the literature of solvent stripping for other organic insect.icides such as D D T are, in general, applicable to benzene hexachloride. However, t h e solvent. s e l e c t e d should be stable to sulfuric acid and should not react with aniline. Purified petroleum ether and pentane have been used successfully in this work, Shell pentane being the most desirable because of extremely low aromatic and unsaturate cont,ent. Benzene hexachloride may be estimated in peanuts and peanut products in the following manner: To a 100-gram sample in a Waring Blendor b o d is added 250 ml. of solvent and mixed for 4 to 5 minutes. Then 100 ml. of a 5% (by volume) sulfuric acid solution is added and blended for about 30 seconds. The mixture is t'ransferred to stoppered bottles and centrifuged until a clear solvent layer is obtained (about 5 minutes), a 100-ml. aliquot of which is taken for analysis. In a 400-ml. beaker 50 grams of the acid Celite is mixed with 20 grams of anhydrous sodium sulfate and the mixture is wetted with solvent. The stripping aliquot is added slowly and the mixture stirred with a glass rod [a modification of the Davidow ( 8 ) t e c h n i q u e ] . T h e s l u r r y is thoroughly mixed until the solution is clear and then filtered with the aid of suction through a 60-mm. fritted-glass Biichner funnel. The solid material in the funnel is then
1979
V O L U M E 24, N O , 12, D E C E M B E R 1 9 5 2 washed with two 50-ml. portions of solvent. The filtrate and washings are combined and treated again with about 20 grams of the acid Celite and stirred mechanically for 5 minutes. The solution is filtered with suction through a fritted-glass Buchner funnel and decanted into a 250-ml. separatory funnel, where it is viashed with three 50-ml. portions of distilled water and is shaken 15 seconds each time. The washings are discarded and the solvent is passed through a 20-mm. fritted-glass Buchner funnel containing a 3-em. layer of anhydrous sodium sulfate. (Interfering surface waxes may be removed by chromatographic separation, magnesium oxide-Celite mixture being satisfactory in most instances.) The funnel is then washed with 50 ml. of solvent and the filtrate plus washings is evaporated on a steam bath t o a volume of 5 to 10 ml. The solution is transferred quantitatively to a 16 X 150 mm. test tube fitted with a T 16/12 female joint and, while immersed in a bath of warm 11-ater (40 to 50" C.), is evaporated to dryness under a gentle current of air. Approximately 2 ml. of purified aniline is introduced by means of an automatic pipet and the test tube is fitted with aI; air condenser equipped with a T 16/15 male joint. The aniline is refluxed for 90 minutes and then the contents are allowed to cool to room temperature. I t is essential that the aniline be refluxed vigorously and that condensation occur near the ground-glass joint. The refluxed aniline is then transferred to a 250-ml. separatory funnel viith ca. 50 ml. of ethyl ether, where it is washed with 50 ml. of dilute hydrochloric acid and 50 ml. of distilled Tmter, respectively, being shaken Tvith each for a t least 10 seconds. The washed ethyl et her solution is filtered through a 20-mm. frittedglass, coarse-porosity Buchner funnel containing a 2-cm. layer of anhydrous, ethyl ether-washed sodium sulfate and collected in a 100-ml. beaker containing two glass beads. The funnel is washed with ea. 20 ml. of ethyl ether, and the combined filtrate and washings are evaporated on a steam bath to approximately 1 ml. and h k e n to dryness under a gentle current of air at room temperature. About 0.2 nil. of pentane is added to take up t,he residue, 10 ml. of sulfuric acid-vanadium pentoxide reagent is introduced, and the intensity of the color produced or optical density is read in a spectrophotometer a t 510 mp. For routine analyses a colorimeter using a filter with a transmittance peak between 550 and 570 mp is satisfactory. The Evelyn colorimeter with filter No. 520bi was used by this laboratory in obtaining the data shown in Tables I, 11, and 111. When most fruits and vegetables are under examination, unwanted extractives may be readily removed by shaking or stirring the solution with 5 to 10 grams of the acid Celite for a feTv seconds and then filtering. Essentially the same procedure may be employed for the analysis of other materials, except in cases where large amounts of surface waxes are encountered and special clean-up techniques are needed. STANDARD CURVES
Because the reaction products of benzene hexachloride and aniline vary when large and small amounts of benzene hexachloride are used, standard curves for both the gamma isomer and technical benzene hexachloride must be prepared by carrying known amounts (0, 20, 40, 60, and 100 micrograms) of each thiough thP entire procedure. The 0 sample or reagent blank
is used as a reference standard when reading unknowns. The slopes of standard curves may vary slightly wit,h changes of reagents and, therefore, should be checked frequently. Recoveries may be checked by adding known amounts of benzene hexachloride to previously analyzed samples and carrying them through the procedure. DISCUSSION
The dichlorodiphenylamine forming a purple color with a maximum absorption a t 555 mp on oxidation by the vanadic acid was obtained from the ether-soluble reaction products by vacuum distillation. Klein ( 4 ) obtained 22 grams of amber colored liquid, boiling a t 145' C. a t 2-mm. pressure, from 40 grams of material produced by refluxing 150 grams of gammabenzene hexachloride with 350 ml. of redistilled aniline and separating the resulting mixture as indicated in the procedure. Total chlorine analysis indicated the amber colored liquid to be a dichlorodiphenylamine. Crystals obtained from this liquid after long storage (ea. 5 weeks) at -30" C. were recrystallized from hexane and melted a t 63.0" to 63.2" C. Orange crystals, which formed in the ether-soluble residue after the ether was completely removed, melted a t 139" to 141" C., were insoluble in cold alcohol and hot water, and formed a red color with the acid reagent with a maximum absorption a t 510 mp, ACKNOWLEDGMENT
The author is indebt'ed to the Dow Chemical Co., Hooker Electrochemical Co., and E. I. du Pont de Xemours & Co., for their generous contributions of chemicals necessary for this v,-ork and to A. K. Klein, Food and Drug Administration, Washington, D . C.! for his many helpful suggestions and appreciation t o Margaret French and Mary Wilson for their assistance in performing the large number of analyses required in carrying out this project. LITERATURE CITED
(1) Bowen, C. V., J . Assoc. O&. A g r . Chemists, 33, 7i4-82 (1950). ( 2 ) Davidow, B., Ibid., 33, 130 (1950). 13) Fairing, J. D., and Phillips, TV. F., Division of Agricultural and Food Chemistry, Symposium o n Methods of -4nalysis for Micro Quantities of Pesticides, 119th Meeting, AM. CHEM. Soc., Boston, Mass. ( 4 ) Klein, A. K.. Food and Drug Administration, Kashington, D . C., private communication. (5) Kolthoff, I. RI., and Noponen, G. E., J . Am. C h e m . SOC.,55, 1448 (1933). (6) Meunier, &I. J., Am. Chem. (61, 1 0 , 2 2 3 (1887). (7)Schechter, M. S., a n d Hornstein, InVin, i l 6 . i ~CHEM,, . 24, 544 (1952). ( 8 ) Slade, R. E., Chemistry and I n d u s t r y , 6 4 , 314 (1945). (9) Wieland, H., Ber., 46, 3296 (1913); 52, 886 (1919). for review May 16, 1952. RECEIVED
Accepted September 13. 1932.
