Colorimetric Determination of Small Quantities of 1,1,1-Trichloro- 2,2

Colorimetric Determination of Small Quantities of 1,1,1-Trichloro2,2-bis(p-methoxyphenyl)-ethane J O H N D. FAIRING and HORACE P. WARRINGTON, JR.

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In a sensitive and specific colorimetric method 1,1,1 -trichloro-2,2bis(p-methoxyphenyl)-ethane is extracted from plant or animal tissue, using benzene or petroleum ether as the solvent. The solvent is evaporated at room temperature by a current of air and the residue dehydrohalogenated with 2 % alcoholic potassium hydroxide. By petroleum ether extraction the resulting 1,1dichloro-2,2-bis(p-methoxyphenyl)-ethylene is removed from the reaction mixture. After the solvent is removed by air evapo ration the dehydrohalogenated methoxychlor is isolated from the nonsaponifiable portion of the fats and waxes by dissolving the residue in hot acetone, chilling, and filtering. After the acetone is removed by air evaporation, the residue is treated with 8 5 % sulfuric acid. This produces a red solution with an absorption maximum at 555 mμ, the intensity of which can be read on a colorimeter and is a function of the methoxychlor concentration. Beer’s law is obeyed over the range of 1 to 50 micrograms.

A need has arisen for a sensitive and specific method of estimating microgram quanti ties of the insecticide methoxychlor i n plant and animal products. Methoxychlor, l,l,l-trichloro-2,2-bis(p-methoxyphenyl)-ethane, is an analog of D D T and therefore procedures for the determination of D D T should be applicable to methoxychlor as well. Such has actually been found to be the case. The pyridine-xanthydrol method of Stiff and Castillo (4) and Claborn (1) will i n d i cate the presence of methoxychlor, but it has a low order of sensitivity and does not distin guish between methoxychlor and D D T . M u c h more promising is the method of Schechter and Haller (3), which depends upon the coupling of the tetranitro derivative of D D T with methanolic sodium methylate. Under conditions of intensive nitration methoxychlor will yield b y this procedure a red color with an absorption maximum at 535 πιμ. It may be generally stated that the Schechter-Haller procedure will distinguish methoxychlor from ρ , ρ ' - D D T , because the latter compound gives a blue color. H o w ever, ο,ρ'-DDT and various breakdown products of D D T yield red colors with absorption maxima close to that of methoxychlor, so that for practical purposes it is impossible to distinguish between them. It appears that the greatest value of the Schechter-Haller method applied to the determination of methoxychlor is in the analysis of materials whose previous spray his tory is unknown. The production of a red color i n such analysis indicates the possibility of the existence of methoxychlor, and warrants a further specific test. Upon treatment with alcoholic potassium hydroxide, methoxychlor will undergo 260


FAIRING AND WARRINGTON—COLORIMETRIC DETERMINATION OF METHOXYCHLOR

261

quantitative dehydrohalogenation. The dehydrohalogenated p r o d u c t reacts with 8 5 % sulfuric acid to produce a red complex of unknown c o m p o s i t i o n w i t h an absorption maximum at 555 ηιμ (Figure 1). No other organic insecticide now i n use produces any color under similar conditions. Therefore, the method is specific for methoxychlor. Fats and waxes, however, yield strong brown colors which will completely mask the methoxychlor reaction. I n the method described this interfer ence has been reduced to a point where it introduces an error of less than 1% when the methoxychlor concentration is between 5 and 50 micrograms. Quantities of methoxy chlor of less than 1 microgram may be determined b y this method. There are six steps involved i n the procedure: 1. Extraction of the methoxy chlor from the material under ex amination with a suitable solvent. ~3S> 4O0 3 5 i o ô s S e o To 2. Dehydrohalogenation with WAVELENGTH- MLLIMICRONS alcoholic potassium hydroxide. 3. Separation of the dehydro Figure 1. Absorption of Red Complex halogenated derivative from the reac tion mixture with petroleum ether. 4. Further isolation of this material from the nonsaponifiable portion of fats and waxes with acetone. 5. Reaction of the dehydrohalogenated methoxychlor with 8 5 % sulfuric acid. 6. Spectrophotometry measurement at 555 ηιμ of the optical density or per cent transmittance of the resulting color. I n order to prepare the methoxychlor for analysis, it must be removed from the m a terial under examination b y means of a solvent extraction. The procedures used for the stripping of D D T are applicable to methoxychlor (2, 5, 6). The solvent chosen should readily dissolve methoxychlor, be sufficiently volatile to be evaporated b y an air current at room temperature, and leave no residue upon evaporation that will interfere with the analysis. Benzene and petroleum ether satisfy these requirements rather well. I t is, however, essential that the solvents be absolutely pure. Reagent grade or C.P. chemicals are usually adequate for the initial extraction, but each batch should be checked to see that i t does not introduce an error into the determination. The extracted methoxychlor is isolated by evaporating the solvent with a current of air. N o measurable loss of methoxychlor has been found when this evaporation takes place at room temperature. However, precautions should be taken to avoid prolonged exposure to the air stream after the solvent has evaporated. Dehydrohalogenation is readily accomplished b y heating with 2 % potassium h y droxide i n 9 5 % ethyl alcohol. I n order to separate the dehydrohalogenated methoxychlor from the reaction mixture, the alcohol is allowed to evaporate, and the residue is taken up i n petroleum ether and washed with water. M o s t batches of even reagent grade petroleum ether contain substances which after contact with potassium hydroxide will yield


262

ADVANCES IN CHEMISTRY SERIES

colors with sulfuric acid. I t is therefore essential to purify the petroleum ether used for this extraction. It is necessary to isolate the dehydrohalogenated methoxychlor from the nonsaponifiable portion of the fats and waxes. T o do this, advantage is taken of the fact that both methoxychlor and its dehydrohalogenated product are readily soluble in acetone, whereas fats are relatively insoluble. The residue is first dissolved i n hot acetone, and then the acetone is chilled to —15° C , which causes precipitation of the fats. After the fats are filtered off, the acetone is removed by evaporation. The intensity of color produced by a given a m o u n t of d e h y d r o h a l o g e n a t e d methoxychlor is dependent upon the acid concentration, as shown i n Figure 2. The optimum concentration is about 82.5%, with the intensity falling off rapidly at lower concentrations. A c i d at a nominal concentration of 8 5 % is used, so that the sensitivity of the reaction is not decreased by the absorption of small amounts of mois ture from the air. I t is difficult to prepare sulfuric acid of an exact concentration and even more difficult to keep such acid at a constant concentration for any length of time. Therefore it is advisable to run a standard of known methoxychlor content along with each set of samples being analyzed in order to correct for changes i n the acid strength. Because Beer's law is obeyed over the range of 1 to 50 micrograms of methoxychlor, i t is not essential to plot more than two or three points on the stand ΊΟΟ 90 80 70 60 ard curve during the daily recheck of the PERCENT SULPHURIC ACIO acid. The color develops slowly with acid of less than 8 2 % strength and not at a l l Figure 2. Effect of Acid Concentra with 6 0 % acid. The addition of water de tion on Color Intensity stroys the color. I n the absence of atmos pheric moisture the color is stable for several days. The reaction rate varies with different batches of acid. W i t h some lots the color is fully developed i n 15 minutes, but occasionally 60 or more minutes are required. A trace of ferric chloride added to the acid assures rapid color development. Analytical Procedure Reagents. Petroleum ether, C.P., boiling range 35° to 60° C ; acetone, C.P., d r y ; a n d ethyl alcohol, C.P., 9 5 % . E a c h reagent is purified before use b y eluting through a column of chromatographic a l u m i n a or b y shaking with activated charcoal and filtering. Benzene, C.P., or petroleum ether, C.P., for stripping. Potassium hydroxide (2%) i n 9 5 % ethyl alcohol, freshly prepared before use. Sulfuric acid, 8 5 % . The actual concentration may range between 82.5 and 8 8 % , but under no conditions should it be less than 82.5%. T o 1 liter of this are added 10 mg. or more of ferric chloride. Sodium chloride, saturated solution. Sodium sulfate, C.P., anhydrous. Standard methoxychlor solution, 10 mg. of recrystallized methoxychlor made up to 1 liter with 9 5 % ethyl alcohol. Standard dehydrohalogenated methoxychlor solution is prepared from 44.71 mg. of recrystallized l,l-dichloro-2,2-bis(p-methoxyphenyl)-ethylene made up to 1 liter with 9 5 % ethyl alcohol. One milliliter of this solution is equivalent to 50 micrograms of methoxychlor.


263

The l,l-dichloro-2,2-bis(p-methoxyphenyl)-ethylene is prepared by refluxing recrystallized methoxychlor i n 9 5 % ethyl alcohol for 2 hours with an excess of potassium hydroxide. It is recrystallized from methanol.


Procedure Extract the methoxychlor from the material under consideration with C.P. grade solvents (benzene, petroleum ether, etc.) that have been checked and found free of interfering substances. Shake stripping with about V a t h of its weight of anhydrous sodium sulfate. A small quantity of some filtering aid may be used if necessary to obtain a clear filtrate, but its use should be limited and it should be checked to see that no loss of methoxychlor occurs with the particular product under analysis. Filter the solution through rapid fluted filter paper. Transfer an aliquot of the filtrate containing between 5 and 50 micrograms of methoxychlor to a 125-ml. standard-taper Erlenmeyer flask. Remove the solvent with a gentle current of dry, oil-free air, preferably at room temperature but never over 35° C . A d d 50 m l . of 2 % potassium hydroxide i n 9 5 % ethyl alcohol. Connect a short length (12 cm.) of glass tubing equipped with a male standard-taper joint to the flask and immerse flask i n a boiling water bath until the volume of the liquid is reduced to about 5 m l . A v o i d prolonged heating after the alcohol has evaporated and run the next step as K> 20 30 40 50 soon as possible. I t is undesirable to hold this alcoholic solution overMICROGRAMS O F METHOXYCHLOR night. Figure 3. Standard Curve Transfer the contents of the flask to a separatory funnel with the aid of 200 m l . of water and 75 m l . of purified petroleum ether. A d d 10 m l . of saturated sodium chloride solution. Shake well, and separate and discard the aqueous layer. Wash two or more times with 200 m l . of water and 10 m l . of saturated sodium chloride solution, discarding the aqueous layers. Filter the petroleum ether extract through 1 or 2 cm. of anhydrous sodium sulfate contained i n a glass Buchner funnel equipped with a 30-mm. fritted-glass plate of coarse porosity. This filtration may be carried out rapidly without the aid of suction. (A small amount of filtering aid may be mixed with the sodium sulfate to remove plant pigments.) Wash the separatory funnel with 20 ml. of petroleum ether. Pass this washing through the filter and combine i t with the filtrate. Transfer the filtrate i n portions to a test tube and remove the solvent at room temperature, either with a current of air or by means of reduced pressure. When dry, add 5 m l . of dry acetone and heat from 2 to 4 seconds i n a boiling water bath to effect a complete solution of the entire residue. C h i l l the tube of acetone thoroughly, to —15° C . if possible. A t the same time chill some dry acetone for washing the filter. Filter the cold acetone rapidly into a glass-stoppered test tube b y means of suction through a 2- or 3-ml. micro Buchner funnel, fitted with a fritted-glass plate of medium porosity. Wash the tube and funnel with 3 m l . of cold acetone and combine i t with the filtrate. Evaporate the filtrate with a current of air. A v o i d excessive drying, as it makes the subsequent dissolving of the residue difficult; 0.01 to 0.05 m l . of moisture is frequently desirable. A d d 10 m l . of 8 5 % sulfuric acid (preferably b y means of an automatic pipet).

264

ADVANCES IN CHEMISTRY SERIES

Stopper the tube with a glass stopper and shake well at intervals for 10 minutes. Let the tube stand for 15 minutes to 3 hours. If undissolved particles are i n suspension, filter through a plug of glass wool. This is generally unnecessary. Read the per cent transmittance at 555 τη. i n a colorimeter or spectrophotometer against 8 5 % sulfuric acid. μ


Preparation of Standard Curve B y means of a microburet add 0, 0.1, 0.2, 0.5, 1, 2, and 5 m l . of the standard methoxy chlor solution to a volume of the stripping solvent corresponding to the average size of aliquot taken. Evaporate this mixture to dryness and carry i t through the above pro cedure.. Plot the resulting per cent transmittance semilogarithmically against concen tration (Figure 3). F r o m this standard curve calculate the original concentration of methoxychlor i n the unknown i n parts per million from the formula: W Vo X

WÔVx

=

p

-

p

j

n

'

where W = weight of residue i n micrograms determined from the standard curves, W = weight of sample in grams, V — aliquot of solvent i n m l . , and V = original volume in m l . of solvent used for stripping. x

0

x

0

If a series of determinations is to be made on the same type of material, i t is advisable to make a standard curve by adding known amounts of methoxychlor to a stripping of the material under consideration which is known to be free of methoxychlor. A n y slight variation introduced into the technique b y the presence of other extracted substances will be compensated for i n this way.

Discussion The recovery of methoxychlor has been found to be quantitative from strippings of apples, green beans, peaches, carrots, celery, pears, peas, lamb fat, beef fat, pork fat, and milk. Table I shows the recovery from apples. Table I.

Recovery of Methoxychlor from Benzene Solutions of Apple Wax Methoxychlor Added, y

Methoxychlor Recovered, y

Recovery.

%

0

0

2.5

2.3 2.5 2.9

92 100 116

5.0

4.8 4.8 5.0

96 96 100

10.0

9.8 10.3 10.4

98 103 104

15.0

15.3 15.8

20.0

20.2 20.8

25.0

24.1 24.2

102 105 101 104 96 97

50.0

50.0

100 Av.

100.6

The dehydrohalogenation proceeds rapidly with a yield of better than 9 8 % . The over-all yield of the entire procedure is better than 9 5 % . Because high yields are obtained, it is possible to prepare a standard solution of dehydrohalogenated methoxychlor to be used in the daily recheck of the calibration curve. The use of such a standard greatly reduces the time required and makes possible the frequent checking of the curve at several points with but little additional effort. One gram of methoxychlor is equivalent to 0.8942 gram of the dehydrohalogenated product.



265

In the authors' experience i t has not been possible to obtain a completely colorless blank, for even the most carefully purified reagents will yield a light yellow color. H o w ever, this color has no effect upon the determination of methoxychlor, for if the solvents have been properly purified this reagent blank will not lower the transmittance at 555 ηιμ b y more than 0.25%. E v e n with the best technique, however, some interference will still be encountered from fats and waxes. W i t h the acetone separation method described this interference is reduced to a point where it seldom lowers the transmittance by as much as 1% and never by more than 3 % . I t has been found that the off-colors introduced b y any one type of biological material are remarkably constant and i t is therefore thoroughly practical to apply a correction for this interference. The over-all errors caused by solvents and fats reach significant proportions only when determinations are being made at the very limit of sensitivity of this test. The authors have been able to measure less than 1 microgram of methoxychlor with errors not in excess of 15%. For some time the acetone separation step was performed before dehydrohalogena tion. However, great difficulty was experienced i n preventing rather sizable losses, be cause some methoxychlor would dissolve in the precipitated fat. W i t h milk in particular these losses became prohibitive. After dehydrohalogenation most of the fat is saponified and removed, so that only a small nonsaponifiable portion remains to be separated. If any methoxychlor does dissolve i n this remaining fraction it is too small to be measured. Further separation of the methoxychlor from fatty contaminants may be accom plished b y diluting the acetone, after filtering, with an equal volume of water and again filtering. This step is seldom necessary, but when employed i t does provide a means for the removal of the last traces of fatty impurities. Because water will be present, it is necessary to extract the dehydrohalogenated methoxychlor from the solution with petro leum ether. This adds another step to the procedure. Such additional work is hardly warranted. Considerable variation is possible i n the technique of this procedure for the deter mination of methoxychlor. Several steps may be eliminated or shortened for rapid rou tine work, and with suitable biological materials this may be done without appreciably sacrificing the accuracy of the test.

Acknowledgment The authors wish to thank Ε. I . du Pont de Nemours & Company for supplying the samples of rriethoxychlor used i n this work, and M a r y L . Wilson for carrying out much of the routine experimental work.

Literature Cited (1) Claborn, H . V., J. Assoc. Offic. Agr. Chemists, 29, 330 (1946). (2) Fahey, J . E., Cassil, C. C., and Rusk, H . W., Ibid., 26, 150 (1943). (3) Schechter, M . S., Soloway, S. B., Hayes, R. Α., and Haller, H . L . . Ind. Eng. Chem., Anal. Ed., 17, 704 (1945). (4) Stiff, Η. Α., and Castillo, J. C., Ibid., 18, 316 (1946). (5) Tressler, C. J., J. Assoc. Offic. Agr. Chemists, 30, 140 (1947). (6) Wichmann, H . J., Patterson, W. I., Clifford, P. Α., Klein, A . K . , and Claborn, Η. V., Ibid., 29, 188 (1946).

Colorimetric Determination of Small Quantities of 1,1,1-Trichloro- 2,2

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