SEPTEMBER 15, 1935
AWALYTICAL EDITION
The grinding chamber is made of a steel tube 3.25 inches in diameter by 3.75 inches long, machined on the inside and ends, as illustrated. The ends are steel plates 3.5 inches in diameter bv 0.25 inch thick, turned and grooved t o fit the chamber. A hble was drilled and threaded in the center of one plate to fit a 0.375- by 4.5-inch steel rod threaded at both ends. The center of the other plate was drilled with a slightly larger hole. A nut was screwed on the outside of the rod, which runs through the center of the chamber, tightening the two plates against the turned steel tube. The plates and steel tube should be machined carefully; otherwise the mill will not retain finely divided material. These specifications do not have to be closely adhered to, but may be varied as needed.
343
abraded during the process, etc.) the type of chamber may be modified. In certain cases, ordinary rubber or cork-stoppered round bottles, from 3 to 5 inches in diameter, with glass marbles about 0.75 inch in ,diameter, were used advantageously, as in the pulverizing of bacteria, sugars, and plant materials. Uniform mixtures of soil or semi-plastic suspensions have been produced by subjecting the material to the rolling process in bottles, without the aid of marbles or balls. When larger scale methods are advantageous, a series of roller units may be constructed one upon the other, connected to a single motor.
The rate of grinding and the degree of subdivision depend upon the interrelation of the amount of material inserted, the size and speed of mill, and the number, size, and weight of balls employed. It was found that 0.75-inch steel ball bearings were most generally efficient. Depending upon the materials ground and requirements conditioned by the investigation (freedom from impurities
Acknowledgments The writer wishes to express his thanks to members of the staff of the Division of Plant Nutrition and to V. Arntaen of the Department of Engineering for suggestions given. RECEIVED JuIy 16, 1935.
Constituents of Pyrethrum Flowers J
Determination of Pyrethrin I1 H. L. HALLER AND FRED ACREE, JR. Bureau of Entomology and Plant Quarantine, U. S. Department of Agriculture, Washington, D. C.
S
IKCE the isolation and characterization by Staudinger and Ruzicka (IO) of the insecticidal constituents of pyrethrum flowers, pyrethrin I and pyrethrin 11, nine methods have been proposed for their quantitative determination. Three of these methods determine pyrethrin I and pyrethrin I1 separately; the other six determine the total pyrethrin content. Staudinger and Harder (9) were the first to propose a method for determining the pyrethrins separately. Their method, known as the acid method, is based upon the fact that both the pyrethrins are esters and that on hydrolysis pyrethrin I yields a monocarboxylic acid volatile with steam, whereas pyrethrin I1 yields a dicarboxylic acid which is nonvolatile. The methods of Tattersfield, Hobson, and Gimingham (11) and of Seil (8) are modifications and improvements of the original Staudinger and Harder method. Of the three procedures, that of Seil is the most rapid, and is probably the most satisfactory. A11 three methods tend to give high results because of the presence of fatty acids, which are known to be present in pyrethrum extracts both in the free state and in combination as esters or glycerides ( 7 ) . If the two pyrethrins were equally toxic to insects, it mould be immaterial which method of analysis was used, but most tests indicate that pyrethrin I is more toxic than pyrethrin 11. Determination of the individual pyrethrins, therefore, may give a more accurate insecticidal value of pyrethrum flowers than a determination of the total pyrethrins. It is well known that most methyl esters yield methyl iodide quantitatively on refluxing in constant-boiling hydriodic acid (5, 6). The method proposed in this paper is based upon the fact that pure pyrethrin 11, being a methyl ester, yields, on refluxing with hydriodic acid, the quantity of methyl iodide required by its formula (4). The latter is determined by the volumetric method of Viebock and Schwappach as modified by Clark (S). I n this method the methyl iodide is absorbed in a n acetic acid solution of potassium acetate to which bromine has been added. The following reaction then takes place:
++ +
+
CHJ Brz -+-CH3Br IBr IBr 2Brz 3H20 +HIOa
+ 5HBr
The solution containing the iodic acid is treated with formic acid to remove the excess bromine, potassium iodide is added, the solution is acidified with dilute sulfuric acid, and the liberated iodine is titrated with a standard sodium thiosulfate solution. As 6 atoms of iodine are liberated for each mole of methoxyl (OCH3), 1 cc. of 0.05 N sodium thiosulfate is equivalent to 3.11 mg. of pyrethrin 11. It thus follows that, for pyrethrum flowers containing about 1 per cent of pyrethrins, a 5-gram sample is ample for a macrodetermination. The determinations were made in the apparatus described by Clark (2).
Reagents The following reagents, all of analytical grade, were used in the determinations, and it was considered essential to determine the blank on them: Petroleum ether (b. p. 30" t o 60" C.) Chloroform Phenol Hydriodic acid, sp. gr. 1.70, constant-boiling, which has been treated with hypophosphorous acid t o remove the free iodine. (The hydriodic acid furnished by Merck & Co. has been treated with hypophosphorous acid.) In order to reduce the blank, it is desirable to pass a stream of carbon dioxide through the boiling solution under reflux for 2 or 3 hours. Potassium acetate solution, 20 grams dissolved in 200 cc. of glacial acetic acid Sodium acetate solution,. 25 grams dissolved in 100 cc. of water Bromine Formic acid (at least 90 per cent purity) Sulfuric acid solution, 10 cc. dissolved in 100 cc. of water Potassium iodide ' Sodium thiosulfate solution, 0.05 N
Procedure PREPARATION OF EXTRACT.A 5-gram sample of finely ground pyrethrum flowers was extracted for 7 hours with petroleum
1NDUST.RIAL AND ENGINEERING CHEMISTRY
344
ether (b. p. 30" to 60' C.) either in a Soxhlet or in a Butt extractor ( 1 ) . For the Soxhlet extraction a 100-cc. extractor fitted with an extra-length condenser was used. The Butt extractor used was 18 cm. long and 2.5 cm. in diameter. It was fitted with an Allihn condenser and provided with a ground-glass joint in order that the same flask might be used for the extraction and the methoxyl determinations, making the transfer from one flask to another unnecessary. The sample for the Soxhlet extractor was handled in the usual manner; that for the Butt extractor was wrapped in filter paper
TABLE I. VALUES FOR PYRETHRIN 11 OBTAINED BY THE &fETHOXYL METHOD, AS COMPARED WITH VALUES OBTAINED BY THE ACID METHODS Powder No. 1932-1
Type of Extractor
Time of Pyrethrin I1 Found Boiling in Meth- TattersMethoxyl oxy1 field Seil Detn. method method method Hours % 9% %
Butt Butt Butt Butt Butt Soxhlet Soxhlet Soxhlet
2.0 2.0 1.25 1.25 1.25 1 25 1.25 1.25
0.38 0.38 0.37 0.37 0.37 0.39 0.39 0.37
0.46 0.42
, . . .
,...
Butt Butt Butt Soxhlet Soxhlet
1.75 1.75 1 75 1 5 1.5
0.33 0.34 0.34 0.36 0.34
0.43 0.42 0 43
0.40 0.39
, . . .
....
1934-2"
Soxhlet Soxhlet
1.5 1.5
0.25 0.23
0.30 0.29
0.28 0.30
1934-3
Soxhlet Soxhlet
1.5 1.5
0.37 0.37
0.52 0.53
0.47 0.47
1934-4 b
Soxhlet Soxhlet Soxhlet Soxhlet Soxhlet Soxhlet
1.0 1.0 1.0 1.5 1.5 1.5
0.35 0.37 0.36 0.39 0.36 0.88
0.47 0.47 0.48
0 54 0.54
0.28 0.28
0.32 0.33
, . . ,
1.0
0.22 0.22 0.23 0.23
,...
,
, . . .
1.5 1.5
0.11 0.11
0.14
....
(18).
PYRETHRIN I1 DETERMINATION. After the extraction most of the solvent was removed by heating in a water bath a t 70 C. When the extract was prepared in the Soxhlet extractor, the residue was quantitatively transferred to the flask used for the methoxyl determination by means of 20 cc. of chloroform used in small portions. A small boiling rod was then placed in the flask and the chloroform was removed by heating in a water bath, the last traces being removed under reduced pressure. Two and one-half cubic centimeters of melted analyticalquality phenol and 5 cc. of constant-boiling hydriodic acid were added, and the flask was connected with the remainder of the apparatus. The absorption tubes contained 10 cc. of potassium acetate solution, to which 20 drops of bromine had been added. Unless a large excess of bromine is used, low results are obtained. The mixture in the flask was boiled at such a rate that the vapors did not rise more than half the length of the condenser, and a t the same time a slow stream of carbon dioxide was passed through the flask. After 1.5 hours the boiling was discontinued, and the contents of the absorption tubes were washed into a flask containing 5 cc. of sodium acetate solution. The volume of liquid was adjusted to about 125 cc., and 20 drops of 90 per cent formic acid were added to remove the excess bromine. When the liquid had become water-white and the last traces of bromine had been removed, 2 cc. of dilute sulfuric acid were added, followed by 1 gram of potassium iodide. The free iodine thus liberated was titrated with a 0.05 N solution of sodium thiosulfate, I cc. of which is equivalent to 3.11 mg. of pyrethrin 11. It thus follows that, for a 5-gram sample:
T-OL. 7 , NO. 5
1934-1
10
Russian Roseum
Butt Butt Soxhlet Soxhlet Soxhlet Soxhlet
1.0 1.0
1.0
, . . . , , . .
0.42 0.42
.... .... , . . .
.. .
... ...
.
I
.
.
I . . .
.... ..
O,l4
...
....
....
,..
,...
,... , . . .
....
a Time of extraction S hours.
b 10-grain samples were used and the time of extraction range:! from 10 to 18 hours.
Summary Cc. of 0.05 N sodium thiosulfate X 0.0623 = per cent pyrethrin I1
Results Table I shows results obtained by the proposed method as compared with values found by the Tattersfield, Hobson, and Girningham method and the Seil method. In all cases the values obtained by the proposed method are somewhat lower than those obtained by the other two methods. The greatest difference occurs in powder No. 19344, although larger samples (10 grams) and longer periods of extraction (10 to 18 hours) were used. As the presence of free fatty acids, esters, and glycerides in the flowers tends to give high results with the acid methods, the values obtained by the methoxyl method probably more nearly represent the true pyrethrin I1 content. Since variation in the time of boiling from 1 to 2 hours made little difference in the results, 1.5 hours was adopted as the recommended procedure. If pyrethrum flowers contained appreciable quantities of methyl pyrethrolone, as is claimed by Ripert ( 7 ) , the results obtained by the methoxyl method would be significantly higher than the values obtained by the acid methods, since methyl pyrethrolone is soluble in petroleum ether and has a methoxyl content about twice that of pyrethrin 11. The results obtained on extracts made in a Butt-type extractor are about the same as those obtained on extracts prepared with a Soxhlet extractor. The Butt extractor has the advantage of requiring less solvent for extraction and also of eliminating the necessity of transferring the extract from the extraction flask to the methoxyl flask. Pyrethrum powder KO.10 is about 10 years old. The analyses obtained by the acid methods were made in December, 1934, and those by the methoxyl method in February, 1935.
A rapid method for the determination of pyrethrin 11, based on the fact that pyrethrin I1 yields methyl iodide when boiled with hydriodic acid, is proposed. The methyl iodide is determined volumetrically by converting it into iodic acid and titrating with sodium thiosulfate the iodine liberated 011 addition of potassium iodide. A comparison of the results obtained by the proposed method with values obtained by the acid methods shows that in all cases the new method gave values somewhat lower than the acid methods.
Acknowledgment The writers wish to express their appreciat'ion to J. J. T. Graham, of the Food and Drug Administration, U. S. Department of Agriculture, for supplying the samples of pyrethrum powders and the results that he obtained by the acid methods.
Literature Cited (1) Butt, C. A,, J. IXD. EXG.& E X , 7, 1930 (1915). (2) Clark, E. P., J. Am. Chem. Soc., 51, 1479 (1929). (3) Clark, E. P., J . Assoc. Oficial A g r . Chem., 15, 136
