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microgram lead standard, and for curve B a blank dithizone solution (15 ml. of a solution containing 4 mg. of dithizone per liter) would be appropriate. If the Photelometer reading for these reference solutions deviates from the original Calibration curves, all abscissas of the points on the curves should be displaced in the same direction an equal distance over the useful range of the curves. This shifting of the curves is legitimate as the useful portions of all curves are substantially parallel.
Acknowledgment The writer is indebted to members of the staff of this laboratory for their constructive criticisms. Literature Cited (1) Assoo. Official Agr. Chem., “Official and Tentative Methods of Analysis”, p. 375 (1935). (2) Clifford and Wichmann, J. Assoc. Oficial Agr. Chem., 19, 132 (1936). (3) Ibid., p. 146. (4) Hoffman, J. Bid. Chem., 120, 51 (1937). (5) Sanford, Sheard, and Osterberg, Am. J. Clin. Path., 3 , 4 0 5 (1933).
FIGURE1. LEAD-DITHIZONE SYSTEM The concentrations of dithizone for cufves A , B , C, D , and E are 2.0, 4.0, 8 0 12.0 and 18.0 mg. per liter, respectively. A different lead standard was iseh ,for‘ each point: the circles and croeses indicate different dithirone solutions. Filter, 610 my, 1-om. absorption cell. Volume of dithirone solution, 15 ml.
range from 0 to 15 micrograms and curve D is suitable for the region from 15 to 50 micrograms. Although Clifford and Wichmann specify a blue-green filter for the lead-dithizone system, they make the following statement ( 3 ) : “Theoretically a better spread could be obtained by working a t 610 mp but the mechanics of the reaction require a small excess of dithizone to be present even a t the upper end of the range to hold the lead as PbD2 in the chloroform phase. Under these conditions the so-called ‘saturated’ color takes the form shown by the dotted line, the transmission being greatly repressed by this excess. Transmission in the green is little affected.” The importance of an excess of dithizone to prevent the decomposition of the red lead dithizone a t the upper end of the lead ranges is recognized. However, the useful range of the calibration curves of Figure 1 obtains for solutions in which an excess of dithizone exists. The upper limit of the useful range of curve A corresponds approximately to a transmission factor of 0.97 (Photelometer reading = 97.0) ; curve B, approximately 0.95; curve C, about 0.92; etc. Above these transmission factors where the excess of dithizone disappears because of its reaction with the greater quantities of lead, the decomposition of lead dithisone probably occurs on account of the equilibrium P b 2D $ PbD2. Thus, the instability of the complex lead dithizone occurs in the insensitive and unused portion of the calibration curve. The precision of the instrument for the lead analysis may be determined from the curves of Figure 1. For the range of 0 to 15 micrograms of curve B, the readings cover twenty-two scale divisions which can be estimated within 0.2 division without difficulty and to 0.1 division by careful observers. This condition indicates a precision greater than 1 per cent. Thus, the precision of the instrument exceeds the accuracy of the chemical procedure even for this lower lead range. Because of the possible instability of chloroform-dithizone solutions or the probable difficulty of preparing an exact duplicate of the original chloroform-dithizone solution, it is advisable to check each calibration curve frequently, using in the procedure a standard lead solution equivalent to the lead concentration a t the lowest portion of the useful range of the curve. For curve D this standard solution would be a 15-
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A Method for Determining Deguelin in Derris and Cube LYLE D. GOODHUE AND H. L. HALLER Bureau of Entomology and Plant Quarantine, United States Department of Agriculture, Washington, D. C.
HEN derris or cube is extracted with a suitable solvent, such as chloroform, the resulting extractive can easily be divided into three fractions. The rotenone can be removed by crystallization from carbon tetrachloride (9, IO), and the remaining resin can be separated into an alkali-soluble and an alkali-insoluble portion (5). The last part has been assumed to consist largely of optically active deguelin, since racemic deguelin is often readily obtained on further treatment of this fraction with dilute alkali. The insecticidal effect of the alkali insoluble fraction, which contains optically active deguelin, has been shown to be of the same order as that of rotenone (1.2). I n its optically inactive form, however, deguelin and its dihydro derivative are much less toxic than rotenone, except when applied in kerosene-cyclohexanone solution (2,IW). Since the toxicity of the noncrystalline fraction may at times be due largely to the presence of optically active deguelin, a further study of the methods for determining this compound seemed desirable. By subtracting that portion of the dehydro compound which results from rotenone, deguelin can be estimated by the method of Takei ( I S ) or Tattersfield’s modification (14),which depends on the formation of the dehydro compounds. Other materials similar to rotenone and deguelin would also be expected to form dehydro compounds, and the results by this method are probably too high. Cahn, Phipers, and Boam ( I ) report a method for the determination of deguelin that is based on the Goodhue modification (3)of the Gross and Smith red-color test. By this method they find the amount of deguelin in a given type of derris resin to be substantially constant. Recently the writers have encountered many samples which appear to have considerable deguelin when tested by both the red-color analysis and the dehydro-compound
DECEMBER 15, 1939
ANALYTICAL EDITION
method, but which give little or no crystalline material upon racemization with dilute alkali. This led them t o suspect the presence of other compounds, and consequently to question the results for deguelin obtained by those methods.
Experimental Method In a recent paper Goodhue and Haller (4) have shown that inactive deguelin forms stable solvates in the same manner as does rotenone, and the procedure described here is based on a method of separation of the desired substance in the form of the carbon tetrachloride solvate which can be conveniently weighed. An extract of the ground material is freed of aqueous alkali-soluble substances and rotenone, leaving the resin containing the deguelin. The resin is then treated with dilute alcoholic alkali to change the deguelin into the crystalline racemic form, which is purified by crystallization from carbon tetrachloride and weighed as the 1 t o 1 solvate. The purity is determined by the red-color test. MATERIALS.The analyses were made on seven samples of derris, five of cube, and one of timbo. The derris was for the most part taken from finely ground commercial samples. The variation from sample to sample was expected to be great, since commercial derris is obtained from a number of species and varieties. Two authentic samples of Derris elliptica, 4175 and 4176, and one sample of Derris malaccensis 4177, were obtained from the Department of Agriculture of the Federated Malay States and Straits Settlements. The samples of cube and timbo (Lonchocarpus) were obtained from commercial sources. PROCEDURE. Fifty grams of finely ground material that contains deguelin are extracted with chloroform in a Soxhlet for 7 hours. Nearly all the chloroform is evaporated on the steam bath, and the extract is taken up in about 75 cc. of ether. The ether solution is extracted with two 15-cc. portions of 5 per cent potassium hydroxide saturated with sodium chloride. The two portions of alkali are extracted with ether, and this ether, combined with the first portion, is washed once with 1 to 10 hydrochloric acid and once with a saturated sodium chloride solution. The alkali-soluble extract is diucarded. The ether is removed on the steam bath, and the resin is taken up in 40 cc. of carbon tetrachloride. This solution is seeded with
A method for isolating and determining deguelin in derris and cube is proposed. After the rotenone and the aqueous alkalisoluble materials have been removed, the remaining resin is treated with dilute methanolic alkali. The resulting racemic deguelin is crystallized from carbon tetrachloride and weighed as a 1 to 1 solvate. The purity of the solvate is determined by the Goodhue red-color test. The amount of deguelin in different samples of derris and cube varies greatly. Eight out of the thirteen samples that were analyzed contained less than 1 per cent. One sample of derris contained 3.9 per cent, and one gave the low value of 0.24 per cent. The samples of cube examined varied in deguelin content from a high of 2.3 to a low of 0.25 per cent. The high toxicity to insects of the noncrystalline portion of derris and cube extracts, coupled, with a generally low deguelin content, suggests the presence of other unidentified compounds that contribute to the toxicity.
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the rotenone-carbon tetrachloride solvate and allowed to crystallize overnight at 0' C. The solvate is then filtered and washed twice with 10-cc. portions of ice-cold carbon tetrachloride. This solvate may be used for the determination of rotenone if desired. The filtrate is evaporated on the steam bath to remove the carbon tetrachloride and taken up in 10 to 15 cc. of methanol. This solution is put while warm in a 25-cc. Erlenmeyer flask, and 10 drops of 40 per cent potassium hydroxide are added. The contents are swirled to mix, aud the flask is carefully filled with warm methanol. A one-hole stopper carrying a funnel made from a drawn-out test tube is immediately inserted. If the stopper is inserted pro erly, no air bubbles remain in the flask and some of the colorless [quid is forced up into the funnel. More methanol is poured in the funnel to take care of the contraction of the liquid on cooling and as a reserve for evaporation. The methanol solution should be kept at about 45' C. for an hour to prevent separation of resin before it is racemized. If deguelin is present, crystals soon separate, but racemization is usually not complete until the material has stood overnight. The flask of racemized deguelin is cooled at 0' C. for 1 hour. The methanol is then decanted through a small filter and allowed to drain as completely as possible without being wauhed. For purification the deguelin crystals are dissolved in a little chloroform, and the chloroform is replaced by evaporating t o a thick solution twice with carbon tetrachloride. Finally the deguelin is crystallized from either 5 or 10 cc. of carbon tetrachloride, depending on the amount present. I t is usually necessary to seed the solution with the carbon tetrachloride solvate of deguelin at 0' C. and let it stand overnight for complete crystallization. The crystals are then filtered on a tared Gooch crucible, washed with cold carbon tetrachloride saturated with deguelin, air-dried at room temperature for 4 hours, and weighed as the 1 to 1 deguelincarbon tetrachloride solvate. The amount of deguelin in the impure solvate is determined by the red-color test ( 3 ) . It is assumed that deguelin alone is responsible for the color, and the fact that racemic deguelin gives only 80 per cent of the color of rotenone is taken into consideration when rotenone is used as the standard of comparison. The solubility of racemic deguelin in methanol and carbon tetrachloride was determined at 0," C. Saturated solutions were prepared at 40" C., cooled t o 0 , and seeded. After standiiig overnight, they were filtered by gravity in the cold room, and the amount of deguelin was determined by the Goodhue red-color test, The carbon tetrachloride solution contained 0.25 gram and the methanol solution 0.11 gram of deguelin per 100 cc. In calculating the results, the effect of these solubilities is compensated for by adding 0.08 per cent when only 5 cc. of carbon tetrachloride are used and 0.11 per cent when 10 cc. are used.
Results The results of duplicate analyses by the racemization method are given in Table I. The deguelin content ranged from 0.25 to 3.29 per cent. A derris of very high rotenone content, sample 4176, contained the most deguelin. Very low results occur among both derris and cube samples. For comparative purposes the amount of deguelin by the red-color test is given for all the samples and the amount by the dehydro method for five samples. I n general the deguelin content is highest by the red-color method; the amount based on the weight of the dehydro compounds is slightly lower, while the results by the new racemization method are the lowest. The amount of deguelin found by this method is extremely variable, even when compared on the basis of the total extractives. The small amount of deguelin obtained by racemization as compared with the amount indicated by the Goodhue redcolor test suggests the presence of other unidentified compounds. Derride ( I I ) , called "elliptone" by Harper (6), is probably responsible for some of the additional red color, but not all, since little or no crystalline material was obtained by racemization. Some of these resins are being fractionated by molecular distillation, and the results of this investigation will be reported later. Discussion of Accuracy While it is difficult t o determine the absolute accuracy of a method of this type, an effort has been made t o check each step and to show that no great error has been introduced.
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ing the carbon tetrachloride crystallization. The amount of resinous substances in the final cry$Deguelin Deguelin Total tallization is not very great and is not expected Carbon in Carbon Deguelina from DeChloroTetra- Tetrachloby RacDeguelin hydro form to have much influence on the solubility of the emisation by Red CornRoteExchloride ride Solsolvate. Sample Solvate vate Method Colorb poundsb noneb traotb Grams % % % % % % An attempt was made to separate the active Derris deguelin from the other resins in derris 4176. 3002 0.6925 61 0.95 4.2 3.1 2.0 12.6 A sample was prepared as outlined above up to 0.6430 59 0.87 59 1.23 3006 0.9463 6.0 4.2 3.6 16.5 the point of racemization and then distilled in 1,2025 58 1.51 51 0.44 3833 0.3505 4.7 ... 4.6 14.0 the high-vacuum still a t 0.0005-mm. pressure. 52 0.46 0.3646 3.3 12.8 The distillate, amounting to 4.1 grams, was dis4174 0.2529 45 0.31 2.6 ... 0.3364 45 0.38 solved in ether to remove any dehydro comDerris elliptica pounds, but none separated. The ether was re4175 1.9420 67 2.71 12.7 ... 6.8 23.4 moved, and the resin was racemized in methanol 67 2.56 1.8540 4176 3.0630 62 3.91 10.3 . . . 11.7 27.0 as usual. A bulky mass of deguelin crystals 2.7640 69 3.92 separated within 10 minutes after the addition Derris malaceensis 5,6 26,3 of alkali, but they were not filtered until the 1.2180 4177 64 1.67 4.8 ... 1.3649 57 1.67 following day. Crystallization from carbon tetCube 8,6 20,0 rachloride gave 1.834 grams of the deguelin sol1.6740 56 1.M 9.0 ... 940A 1.5795 52 1.75 vate, which if pure indicates a deguelin content 40 0.38 3.0 3.1 2.6 16.4 3004 0.3913 of only 2.64 per cent. About 32 per cent of the 40 0.52 0.5538 3005 2.3473 47 2.32 5.8 5.l 5'6 18.4 deguelin was lost in the distillation process. 54 2.16 1.8984 M 1 0.1780 49 0.25 4.0 ... 2.2 13.1 I n the final step, the use of the red-color test, 50 0.27 0.1977 3.9 22.6 eliminates the error due to dehydro compounds M 2 0.2730 54 0.38 6.5 ... 0.5428 45 0.57 and to tephrosin which probably appear in the Timbo 3230 0.5soo 44 0.59 4.1 3, 3, 19, 2 final precipitate. Some degradation products of 0,5316 48 0.59 deguelin or rotenone may also give the red color, a A correction of 0.11 per cent was added for t h e solubility where 10 cc. of carbon tetrabut since by this test the deguelin present in chloride were used for crystallization. Samples obviously containing small amounts, less t h a n 0.6 per cent, require only 5 cc. and thus a correction of 0.08 per cent. the solvate has never been more than 69 per b Some of these values were taken from published (8) and unpublished d a t a b y H. A . cent as compared with the theoretical 71.9, it Jones, of this bureau. appears that little or no material that might add to the color is present. The compound derride (11) should not interfere, since it does not form a solvate with carbon tetraFirst, the possibility that residual rotenone might be carried over was checked by determining the rotation of several of the chloride. After racemization it may cause some discrepancy, but insufficient information about this compound is available deguelin-carbon tetrachloride solvates. Only a slight levoto justify any assumption a t this time. rotation was observed, which indicated the presence in the Finally, in the calculation of the results the solubility of solvate of only about 1 to 3 per cent of its weight, calculated deguelin in both methanol and carbon tetrachloride has been as rotenone. This is considered negligible and might even determined and the appropriate corrections have been made. help to correct for some small negative errors. These amount, a t the most, to only 0.11 per cent. The possibility that much of the deguelin might be destroyed during racemization with dilute alkali was investigated. By high-vacuum distillation a concentrated sample of Precision active deguelh was prepared from cube sample 3005. 'This The precision is about equal to that in the rotenone analygave a red-color value indicating the presence of 79 per cent sis ( I O ) . The agreement is not quite so good, owing to t h e of deguelin, and 50 per cent of the material was separated as large number of transfers and operations that are necessary. the crystalfine Ldihydrodeguelin after hydrogenation. Upon Duplicate analyses should check to about 0.2 per cent. Many racemization, 83 per cent of inactive deguelin was obtained. of the check determinations were made as much as one month The deguelin so obtained melted a t 167" C. (corrected), and apart, and the results were always in good agreement. its crystallographic properties checked those of an authentic sample. Thus it appears t h a t not more than 17 per cent can Literature Cited be destroyed by racemization, and such loss is probably much (1) Cahn, R. S., Phipers, R. F., and Boam, J. J., J . SOC.Chem. Ind., lesfi, since the active deguelin sample used was not pure. 57, 200-9 (1938). To show the contrast with a low-deguelin sample, derris (2) Fink, D. E., and Haller, H. L., J . Econ. Entomol., 29,594-7 3833 was used to prepare a concentrate in the same way. The (1936). red-color test indicated that this sample apparently contained (3) Goodhue, L. D., J . Assoc. Oficial Agr. Chem., 19, 118-20 (1936). (4) Goodhue, L. D., and Haller, H. L., J . Am. Chem. Soc., 61,486-8 60 per cent of deguelin, but no crystalline material could be (1939). obtained by racemization. (5) Haller, H. L., and La Forge, F. B., Ibid., 56, 2415-19 (1934). As in the rotenone analysis ( 7 ) , an excess of resin might (6) Harper, S. H., Chemistry &Industry, 58, 292 (1939). retard or prevent complete crystallization. To show the (7) Jones, H. A., IND.ENG.CHEM.,Anal. Ed., 9,206-10 (1937). (8) Ibid., 11, 429 (1939). effect of time, one sample of derris 4176 was allowed to rac(9) Jones, H. A., J . Am. Chem. Soc., 53, 2738-41 (1931). emize for 3 days and to crystallize from carbon tetrachloride (10) Jones, H. A., and Graham, J. J. T., J . Assoc. Oficial Agr. Chem., for the same time. No difference was noted. This also shows 21, 148-51 (1938). that long contact with dilute methanolic alkali in the absence (11) Meyer, T. M., and Koolhaas, D. R., Rec. trav. chim., 58, 207-17 (1939). of oxygen does not destroy the deguelin. (12) Sullivan, W. N., Goodhue, L. D., and Haller, H. L., Soap, I n many instances some resinous material adheres to the 15, No. 7, 107, 109, 111, 113 (1939). racemized deguelin, and in order to avoid loss the crude (13) Takei, S., Miyajima, S., and Ono, M., Ber., 66,1826-33 (1933). crystalline product is not washed. If the resinous material (14) Tattersfield, F., and Martin, J. T., Ann. Applied B i d . , 22,. 578-605 (1935). clinging to the crystals contains deguelin, it is recovered durTABLEI. DEGUELIN CONTENT O F DERRISAND CUBE BY VARIOUS METHODS
