ANALYTICAL CHEMISTRY
1866 Table I,
Oxygen Content of Sodium at 110’ C.
Sample No. 1 2 3 4
5 6
7 8
9 10
Weight
RESERVOlR
-MERCURY
Y,02
0.002 0.003 0.003 0.003 0.002 0.003 0.003 0.002 0.002 0.003 Av. 0 . 0 0 2 6 Maximum deviation 0,0006
T O V b C U U M 4ND N2 MINlFOLD
‘1019
GLASS
SOCKET
JOINT
CORNING S I N T E R E D ,’ GLASS FlLTER (CCAIISEl
Table 11. Recoveries of Known Amounts of Oxygen Ot Added, Wt. %
01Found, Wt. %
0.037 0.074 0.126 0.151 0.275
0.037 0.075 0.123 0.152 0.280 Mean deviation
Deviation 0.000
+0.001
-0.003 f0.001 +0.005 + O . 002
s
19/38
--_
T O VbCUYM bND MbNlFOLD
of hand magnets.) Add approximately 20 ml. of mercury from the upper reservoir, stirring vigorously with the magnetic slug. (The delivery tube provides a “shower” system which effectively washes the sides of the reaction chamber.) After the amalgam has cooled t o room temperature, open the bottom stopcock and drain all but 1 or 2 ml. of the amalgam into the receiving flask. Add more mercury, stir, and drain until a test of the effluent mercury indicates complete separation of sodium metal and sodium monoxide. Final solution and titration of the sample are identical to the procedure prescribed by Pepkowitz and Judd.
2SO HL F L b S K
~
Figure 1. Apparatus Table I1 gives the results of analysis of samples of the same sodium to which mercuric oxide was added, and which was reduced by heat, This method has been applied \%ithequally accurate results to potassium metal and the sodium-potassium alloys Potassium amalgamates slowly and with some difficulty in the cold, however.
EXPERIMEKTAL
The sodium used in these analyses was cut from Du Pont air-cast bricks and sampled according to the Walters and Miller (8) technique. The molten sodium in the sampling apparatus had a coating of oxide on the surface, indicating saturation with oxide a t the sampling temperature.
LITERATURE CITED
(1) Pepkowitz, L. P., and Judd, W. C., ANAL. CHEM.,22, 1283 (1950). (2) Walters, S. L., and Miller, R. R., Ibid., 18, 658 (1946).
Table I gives the results of the analysis of a series of these samples using the apparatus described.
RECEIVED June 5, 1951.
Colorimetric Determination of Octamethyl Pyrophosphoramide ‘
I
S. A. HALL, J. WM. STOHLILIAN, 111, AND M. S. SCHECHTER Bureau of Entomology and Plant Quarantine, U. S. Department of Agriculture, Beltsuille, Md. T H E increasing experimental use of the new systemic insecticide octamethyl pyrophosphoramide has created the need for a sensitive method for determining minute quantities of it in plant material. Ripper, Greenslade, and Hartley (4)have tested for the presence of the compound by making use of the molybdenum blue method for orthophosphate ion. Their method has been found to suffer interference from phosphorus compounds occurring naturally in plant material. Octamethyl pyrophosphoramide hydrolyzes (4),on heating with strong mineral acid, to yield 4 moles of dimethylamine:
(CH3)aN 0
\I1
(CH,)zN /
P-0-P
0 S(CH8)s H + HsO II/
+4(CHj)2NH + 2H3PO4
‘S(C”3h
A sensitive method for the determination of minute amounts of dimethylamine in biological fluids has been described by Dowden ( 2 ) . His method is based upon the reaction of dimethylamine with carbon disulfide and copper ion to form cupric dimethyl-
dithiocarbamate, which is water-insoluble and imparts a deep yellow color to certain organic solvents in which it is soluble. Dowden chose benzene as the solvent; the authors found that chloroform is preferable and more convenient to use. Although any secondary alkylamine will give Dowden’s color test, Sidgwick (6)has stated that secondary alkylamines are not commonly encountered in biological material. Primary and tertiary amines do not interfere with the color test. The method described in this paper includes chloroform extraction of the sample to be analyzed; acid hydrolysis of the octamethyl pyrophosphoramide in the extractive to dimethylamine hydrochloride; steam distillation with excess alkali, the dimethylamine being absorbed in hydrochloric acid; and development of the color by shaking with alkali, copper reagent, and chloroform containing 0.5% carbon disulfide to produce in the chloroform layer the yellow color of cupric dimethyldithiocarbamate, the intensity of which is measured in a photometer. A typical colored solution was developed from 45 micrograms of dimethylamine hydrochloride (equivalent to 39.5 micrograms of octamethyl pyrophosphoramide) using 5 ml. of chloroform-
1867
V O L U M E 23, NO. 1 2 , D E C E M B E R 1 9 5 1 carbon disulfide. The transmittance-wave length curve (Figure l ) , obtained Lvith a Beckman Model DU spectrophotometer exhibits an absorption maximum a t 435 mp. APPARATUS AND REAGENTS
Erlenmeyer flasks, 125-nil. capacity with T joint to fit reflux condenser, and 25-ml. capacity with T glass stopper. Reflux condensers, West type, 10 em. long. Use Tygon or similar tubing for connecting the water jacket. Glass beads. I
I
zot
'
I
350
Figure 1.
400 450 500 WAVELENGTH, MILLIMICRONS
5
Transmittance-Wave-Length Curve
Separatory funnels, 60-ml. capacity. Make a mark a t the 25-ml. level. I h r i c a t e stopcock and glass stopper _ . with water just before use. Kjeldahl distillation apparatus, 100-ml. capacity, all-glass (no rubber connections). Photoelectric coloiimeter, Klett-Summerson or equivalent. Use No. 42 blue filter (spectral range 400 to 465 mp), Colorimeter tubes, matched. Use corks wrapped with aluminum foil to fit tubes. Blender. Replace rubber g n k e t with Resistofleu. Use aluminum foil on underside of hard-rubber cover. Sodium hydroxide solution, saturated. Hydrochloric acid solution, approximately 1 S. Sodium hydroxide or barium hydroxide, standard solution, 0.02 N . Hydrochloric acid, standard solution, 0.1 N. Copper reagent. Dissolve 0.5 gram of eo per sulfate pentahydrate in 10 ml. of distilled water, and m a i e to 500 ml. xith concentrated ammonium hydroxide (specific gravity 0.9). Chloroform solution of carbon disulfide. Add 5 grams of carbon disulfide to 1 liter of chloroform and mix by shahing. Anhydrous sodium sulfate. Make a small glass scoop to measure about 1 gram for each determination. Dimethylamine hydrochloride, crystalline salt. Antifoam solution. DC Antifoam A (Dow Corning Corp.), saturated solution in chloroform. PROCEDURE
Preparation of Standard Curve. Dissolve 1.63 grams of dimethylamine hydrochloride in 1 liter of distilled water to give a 0.02 N solution. The salt is hygroscopic and cannot be conveniently weighed with accuracy. Determine the exact normality of the solution as follows: Transfer a 25.0-ml. aliquot to the Kjeldahl distillation flask. With the receiving tube of the Kjeldahl dipping into 10.0 ml. of 0.1 N hydrochloric acid (contained in a 125-ml. Erlenmeyer flask) add to the distillation flask about 1 ml. of saturated sodium hydroxide solution and distill. Continue the steam distillation until about 25 ml. of distillate have been collected and then back-titrate with 0.02 N alkali. From the quantity of standard alkali consumed calculate the normality of the dimethylamine hydrochloride solution. Alternatively, if desired, determine the normality of the dimethylamine hydrochloride solution by direct titration of an aliquot with standard 0.02 N silver nitrate solution (chloride ion method).
By appropriately diluting an aliquot of the 0.02 N stock solution of dimethylamine hydrochloride prepare a solution (0.00014 N ) containing 11.4 micrograms per ml. of dimethylamine hydrochloride equivalent to 10 micrograms per ml. of octamethyl pyrophosphoramide. Transfer aliquots to a series of 60-ml. separatory funnels t o contain, respectively 20, 40, 60, etc., up to 200 micrograms of octamethyl pyrophosphoramide equivalents. Make each funnel up to the 25-ml. mark with water. T o develop the color add 5 drops of saturated sodium hydroxide solution, approximately 1ml. of copper reagent, and by pipet 20.0 ml. of chloroform-carbon disulfide reagent and shake for 3 minutes. Draw off the chloroform layer into a 25-ml. Erlenmeyer flask ( T glass stopper), add about 1 gram of anhydrous sodium sulfate, stopper and swirl a few seconds to remove any turbidity, decant the solution into a colorimeter tube, stopper with a foil-covered cork, and measure against a blank sample (run in the same way) in the photometer. The color is stable for a t least 30 minutes. Prepare the standard curve by plotting the logarithm of the transmittance (or the logarithmic photometer reading) against concentration expressed in micrograms of octamethyl pyrophosphoramide. If desired, the standard curve may be based directly on a standard solution in chloroform of pure octamethyl pyrophosphoramide following the procedure described below for the determination of octamethyl pyrophosphoramide in chloroform extracts of plant material. Determination of Octamethyl Pyrophosphoramide in Plant Material. Extract the sample of plant material with chloroform by exhaustive extraction ( 3 or more hours) in a Soxhlet extractor or by maceration of the sample with chloroform in a blender. If a Soxhlet extractor is used, make the extract up to volume with chloroform in a volumetric flask and take an aliquot for the determination. If a blender is used, add a known volume of chloroform to cover the sample, macerate for 20 minutes, and filter a portion of the homogenate through a fluted filter paper. Cover the funnel with a watch glass to minimize evaporation of chloroform during the filtration. Take an aliquot of the filtrate for the determination. If possible, choose an aliquot that will contain from 20 to 150 micrograms of octamethyl pyrophosphoramide. Place the aliquot (5 to 50 ml.) in a 125-ml. T Erlenmeyer flask, add approximately 10 ml. of concentrated hydrochloric acid, drop in a few glass beads, and completely remove the chloroform on the steam bath. Attach a condenser and reflus on a hot plate for 1 hour. Cool, wash down the condenser with a little distilled TT-ater into the acid hydrolyzate in the flask, and transfer quantitatively to the distillation flask of the all-glass Kjeldahl apparatus. Immerse the condensate tube of the Kjeldahl apparatus in about 2 ml. of dilute hydrochloric acid (approximately 1 .V) contained in a 60-ml. separatory funnel serving as the receiver. Then add to the hydrolyzate in the distillation flask a drop of antifoam solution and an excess (about 10 ml.) of saturated sodium hydroxide solution and commence the steam distillation. Collect the distillate in the separatory funnel up to the 25-ml. mark. From this point proceed to develop the color in the manner described above for preparing the standard curve. DI SCUSSIO3
The method of analysis described was developed primarily to determine octamethyl pyrophosphoramide in minute quantities present in plant material as a result of spray or soil applications of the insecticide. Because octamethyl pyrophosphoramide acts as a systemic insecticide and is absorbed by the living plant and translocated in the sap, it is largely contained within the plant and not on the surface. Hence it is essential that plant material containing octamethyl pyrophosphoramide be thoroughly macerated m-ith solvent or exhaustively extracted in a Soxhlet eutractor. In preliminary tests to find the most suitable solvent it vias found that chloroform was the best for this purpose. Benzene, which is commonly used to strip spray residues of D D T , benzene hexachloride, and similar organic insecticides from plant material, was found unsuitable for extracting octamethyl pyrophosphoramide because it gave low recoveries of this insectiride from plant material. Although no interference has thus far been encountered from blank runs on plant material not treated a i t h octamethyl pyrophosphoramide, nevei theless it is always important to run a control analysis on a sample of the untreated material \!-hich is under study. Plant material that has been run by the method includes cotton bolls, peas, pea vines, rose leaves, snap beans, and alfalfa.
1868
ANALYTICAL CHEMISTRY
Table 1. Recover) of Octamethyl Pyrophosphoramide (OIIIPA) from Chloroform Solution Chloroform Evaporated, hll. 100 100 50 50 50 25 25
Micrograms of O M P A I n chloroform Recovered 104 104 52 52
52
26 26
Recovery,
%
95 94 48
47 49 26 24
Av.
91 90 92 91 94 100 92 93
I t is necessary to avoid a t all steps of the analytical procedure any contact with rubber or cork, as these materials introduce yellow colors that interfere and can give high results. On the other hand, chlorophyll and other colored substances extracted from plant material by the chloroform did not interfere in the least with the colorimetric method. The method as described is sensitive to the determination of about 5 micrograms of octamethyl pyrophosphoramide. By appropriate use of smaller volumes of chloroform-carbon disulfide and longer absorption cells, the sensitivity of the method can be increased in the absence of an appreciable blank in the check sample. Where amounts of octamethyl pyrophosphoramide in excess of 150 micrograms are found, using 20.0 ml. of chloroformcarbon disulfide, the yellow color will be too intense to measure accurately in the photometer. I n the latter case one can pipet an aliquot of the colored solution into a small volumetric flask and make up to volume with chloroform-carbon disulfide reagent; the color intensity has been found by this procedure to obey Beer’s law. Purified octamethyl pyrophosphoramide in chloroform s o h tion (1.04 micrograms per ml ) was run through the described procedure. As shown in Table I, an average recovery of 93% was obtained. David ( 1 ) extracted radioactive octamethyl pyrophosphoramide from bean plants by treating the plant material first with 0.2 hr sodium hydroxide solution a t 80’ for 30 minutes and then eytracting with chloroform. The authors tried his procedure on bean plants containing octamethyl pyrophosphoramide. Some difficulty was encountered with emulsions formed during chloroform extraction of the alkaline solution and alkali-treated plant material. The alkali treatment released dimethylamine (derived from octamethyl pyrophosphoramide) which was held in
some form, possibly as a salt, in the plant tissue. No dimethylamine was obtained from control bean plants when subjected to the alkali treatment. In the absence of the alkali treatment it was found that the dimethylamine bound in the plant was not appreciably extracted by chloroform. Analyses obtained by direct chloroform extraction-Le., without preliminary treatment with alkali-showed a general correlation with insect mortality. DuBois and his associates ( 3 )have reported that octamethyl pyrophosphoramide, in its passage through certain animal or plant tissues, exhibits greatly enhanced anticholinesterase effects. The authors confirmed this phenomenon by testing the chloroform extractiveof bean plants, previously treated with octamethyl pyrophosphoramide, for its inhibitory effect on a commercial cholinesterase preparation (purified bovine cholinesterase plus human serum). The enhancement in anticholinesterase effect \vas approximately 700 times that of the original octamethyl pyrophoramide before passage through the sap of the growing bean plant. “Activated” octamethyl pyrophodphoramide as well as the unchanged compound was extractable by chloroform, but the “bound” or metabolized octamethyl pyrophosphoramide (as evidenced by dimethylamine tied up in the plant) was not appreciably removed with chloroform. As the method is based on the determination of dimethylamine, it may not distinguish between octamethyl pyrophosphoramide and chloroform-soluble metabolites containing the dimethylamino group. Kevertheless it appears that direct chloroform extraction of plant material can give a measure of the systemic insecticide. This became evident in experiments in which known quantities of octamethyl pyrophosphoramide were introduced into seedlings of bean plants and the decline of the insecticide was followed as the plants grew to maturity. LITERATURE CITED
(1) David, 11’. A. L., Nature, 166, 72 (1950); also personal communi-
cation.
(2) Dowden, H. C., Biochem. J . . 32,455-9 (1938). (3) DuBois, K. P., Doull, J., and Coon, J. I f . , J . Pharmacal. Erptl. Therap., 99, 376-93 (1950). (4) Ripper, W. E., Greenslade, R. M., and Hartley, G. S., Bull. Entomol. Research, 40 (Part 4 ) , 481-501 (1950). (5) Sidgwick, N. V., “Organic Chemistry of Nitrogen,” p. 21, London,
Oxford University Press, 1937. RECEIVEDNovember 15, 1950. Presented before the Division of Agricultural and Food Chemistry, Symposium on Methods of Analysis for Micro Quantities of Pesticides, a t the 119th Meeting of the .4MERICAN CHEMICAL SOCIETY, Boston, Mass.
Apparatus for Microdetermination of Molecular Weights of Volatile liquids LEONARD K. NASH, Harvard University, Cumbridge,
OUNG and Taylor have described (6) an ingenious modificaY tion of a traditional method for the microdetermination of the molecular weights of volatilizable liquids. The modified method was found to be rapid, capable of extreme sample economy, and accurate to 1 t o 2% in most cases. This otherwise attractive technique involves the use of a rather elaborate micromanometer; and the latter, in turn, entails a commitment t o operations under high vacuum. The very simple apparatus here described retains most, if not all, the favorable characteristics of the Young and Taylor equipment; but the micromanometer and the high vacuum manipulations are entirely eliminated. The ne&-apparatus is based on the use of a click gage to indicate when a certain fiducial pressure difference exists between an external (manometer) system and an internal (testing) system. Newton has shown recently ( 4 ) that the glass membrane system described by Mauquin and Garman ( 3 ) for use as a hydrogen electrode can also be successfully employed as a differential pressure gage, I t has since been found by Bailey (%)that, if an
Mass.
inhomogrneity is introduced into the membrane, it can be operated as a click gage. The instrument so developed is sufficiently sturdy to withstand pressure differentials of 1 atmosphere; yet it is sensitive enough to indicate audibly, without any auxiliary equipment, the existence of a standard pressure differential accurate to 0.1 mm. APPARATUS AND PROCEDURE
The gage based on the design of Mauquin and Garman has a rather awkward geometry, and for the present work the desideratum was a gage with arms disposed rectilinearly. Such a gage may be prepared by following the general directions given by Mauquin and Garman, save that the bulbs are joined end to end, rather than side to side; and two glass-blowing swivels, with leads joined through a T-tube, are attached to the ends of the two arms, so that during the construction of the gage the system can be blown simultaneously on both sides of the membrane. If the membrane so prepared does not click a t one (or more) critical pressure differential(s) it should be wrinkled slightly, by gently heating the gage and blowing first on one and then on the other
,
