Colorimetric Determination of Pyrethrins, Allethrin, and Furethrin CIPRIANO CUETO AND W. E. DALE Bureau of Entomology and Plant Quarantine, U . S. Department of Agriculture, Sawsnnah, Ga. rins concentrate analyzed by the method of the AOAC ( 1 ) . The pretreatment with cold Skellysolve F must be carefully followed before analysis. STANDARD SOLUTION OF PYRETHRINS. 0.1000 gram (based on the AOAC analysis) diluted to 1 liter with ethyl alcohol; 1ml. is equivalent to 0.1 mg. of pyrethrins. S T A S D A R D SOLUTION O F ALLETHRIN. 0.1000 gram Of cr-dltrans-allethrin (crystalline) diluted to 1 liter with ethyl alcohol; 1 ml. is equivalent to 0.1 mg. of allethrin. STAXDARD SOLUTION OF FURETHRIN. 0.1000 gram of furethrin, bayed on 100% pure material, diluted to 1 liter with ethyl alcohol; 1 ml. is equivalent to 0.1 mg. of furethrin.
The use of pyrethrins, allethrin, and possibly furethrin as a coating on paper and cloth bags for the protection of stored food products against insects has created a need for a sensitive, accurate, and rapid method of detection and determination of these materials. Such a method might also find use in the determination of residues on food products and in the analysis of concentrates and various formulations. A colorimetric method developed for the estimation of small amounts of pyrethrins, allethrin, and furethrin is sensitive to about 2007 and has an average experimental error of about 4%. The color reaction on which the method is based can also be used as a color test and can possibly be developed into a colorimetric determination of other cyclic a,B-unsaturated ketones. The method outlined can be used only if it is known which insecticide (pyrethrins, allethrin, or furethrin) is present. Further, if more than one of these insecticides is present it is impossible by this method or any other methodpublishedup to the present time to determine the concentration of each insecticide specifically.
T
APPARATUS
SOXHLETEXTRACTION APPARATUS. All glass connections (condenser, Soxhlet, and 250-ml. Erlenmeyer flask). FUSNELS, 250-ml. capacity. SEPARATORY L4RGE TESTTUBES, about 3 X 20 cm. CYLINDRIC.4L FUNNELS, about 3.5 X 10 cm. Two WATERBATHS,one maintained a t 70" & 5' C. and one at 25" iz 5' C. FILTERPbPER. Reeve h g e l KO.802, 12.5-cm. diameter, folded, rapid-flow, or equivalent. STOPWATCH. Watch with sweep second hand may also be used. COLORIMETER. Klett-Summerson photoelectric colorimeter, Model 900-3, with 12.5-mm. calibrated test tube and green filter having transmittance peak a t 540 mfi. Any comparable colorimeter may be used.
HE AOAC method (1) for pyrethrins and three methods
proposed for the assay of technical allethrin (9) [hydrogenolytic (6), ethylenediamine (5),and polarographic ( 8 ) ] are not applicable or have not been applied to the determination of these insecticides when used in paper coatings. In the first two methods too large a sample of paper would be required. A hydroxylamine method described by Edwards and Cueto ( 3 )is not specific for these insecticides and is applicable only to uncontaminated pyrethrin-coated paper, since fatty acids and esters of the stored food products cause interference in the development of the color, usually giving high results. Furthermore, variations in the ratio of pyrethrin I to pyrethrin I1 introduce variations in the results. A color test for allethrin and pyrethrins reported by Feinstein (4) and a paper by Thompson et al. (IO)describing the reaction of bifunctional sulfur compounds with a,@-unsaturated ketones in the presence of a basic catalyst led the authors to investigate the reaction of sodium sulfide with allethrin. It was found that this reagent gave a color test with allethrin and also with pyrethrins and furethrin. With other cyclic materials having an a,@-unsaturated ketone group, addition of a basic catalyst was required for the development of color. Within the range studied, 0.5 t o 10.0mg., these colorimetric determinations followed Beer's law. REAGENTS
SODIUM SULFIDE REAGEST. Weigh out 1 gram of sodium sulfide pentahydrate (Baker's analyzed reagent) and dilute to 100 ml. with distilled water. Prepare fresh weekly. ACETOXE,C.P. SODIUM CHLORIDESOLUTION.Weigh out 200 grams of ACS grade sodium chloride and dilute to 4 liters with distilled water. SKELLYSOLVE F. A hydrocarbon fraction with boiling range of 200 - - to .- 600 -- c -. ETHYL ALCOHOL, C.P., 95%. HYFLO SUPER-CEL. A diatomaceous filtering aid. COTTON. Absorbent grade, USP. PYRETHRUM CONCENTRATE. Use any standard 207" pyreth-
PROCEDUHE
Preparation of Extract. Cut 0.5 sq. foot of the paper sample into suitably sized strips, place them immediately in the Soxhlet extractor, and extract for 3 hours with approximately 125 ml. of acetone. Without transferring it from the extraction flask, concentrate the extract on the steam bath to a volume of about 50 ml. Transfer the extract quantitatively (rinsing with acetope) to a separatory funnel containing approximately 150 ml. of sodium chloride solution. Add 15 ml. of Skellysolve F. Shake for about 1minute, releasing the pressure a t intervals. Allow the mixture to separate. Draw off the aqueous layer into another separatory funnel and extract in the same manner using 5 ml. of Skellysolve F. Repeat the extraction with another 5-ml. portion of Skellysolve F in a third separatory funnel and then discard the aqueous layer. Combine the extracts, using a minimum of Skellysolve F for transfer into the first separatory funnel. Wash the combined extract with 15 ml. of distilled water and draw off the lower aqueous laver. Allow the extract to drain into a large test tube throuih a cylindrical funnel containing a plug of cotton previously wet with Skellysolve F. Rinse the separatorq- funnel with about 10 ml. of Skellysolve F and pour through thr cotton, allowirig time to drain into the test tube. The volume of the final extract should be about 50 ml. Add a glass bead to the tube and ( w e f u l l y evaporate the solvent from the extract by immersing the tube in a warm-water bath (70" C.), then heat slowly while tapping the tube to initiate bouncing of the glass bead and to ensure smooth boiling of the solvent. Evaporate to dryness. Pipet 10.0 ml. of ethyl alcoliol into the tube and swirl to dissolve the residue. This is a good stopping point. Stopper the tube or proceed directly to development of the color. Development of Color. To the test tube containing the alcoholic solution (10.0 ml.) add by pipet 1.0 ml. of sodium sulfide reagent and mix by gently tapping the tube with the fingers. Observe esact timing from the addition of the sodium sulfide. Do not use a stopper. Place in a water bath (70' f 5" C.). Remove in exactly 15 minutes, add about 0.3 gram of Hyflo-Super Cel, agitate by vigorously tapping the tube, and immerse at once in the 25' =k 5" C. water bath. Twenty minutes after the addition of the sodium sulfide reagent, transfer the contents di-
1367
ANALYTICAL CHEMISTRY
1368 Table I.
Color Reactions Produced by Sodium Sulfide Reagent Compound
Sulfide Reagent Compound Chrysanthemic acid, ester of 2-benzyl-4-hydroxy-3-methyl-2cyclopentene-1-one Chrysanthemic acid, ester of 4-hydroxy-2-phenyl-3-methyl-2cvclopentene-1-one Isophorone Cinerins Allethrolone
Color
Deep red Dark red Dark red Dark orange vermillion
Compounds Which Develop No Color with Sodium Sulfide
Butyl oleate Butyl stearate Casein Chrysanthemic acid Chrysanthemic anhydride Chrvsanthemic dicarboxvlic acid DD? Ethyl laurate Lindane
Methoxychlor Oleic acid Piperonyl butoxide Piperonyl chrysanthemate Sesame oil Starch Stearic acid Sulfoxide (n-octylsulfoxide of isosafrole)
rectly through rapid-flow, fluted filter paper into a calibrated colorimeter tube. Exactly 25 minutes after the addition of the sodium sulfide reagent, take the transmittance reading. iln orange to red color will develop. REAGENT BLANK. Prepare a reagent blank by pipetting 1.0 ml. of sodium sulfide reagent into 10.0 ml. of ethyl alcohol and carrying through the color procedure as described above. No visible color will develop. Make all readings with reference to this reagent blank. CORRECTION BLANKFOR UNTREATED PAPER. Under conditions identical with the treated samples, run a sample of paper of the same origin but not treated with insecticide. The correction blank should be very small-about 1 mg. of insecticide per square foot of paper. Preparation of Standard Curve. Pipet aliquots of the standard solution of the insecticide to be determined into separatory funnels, each containing about 150 ml. of the sodium chloride solution. Use 5, 10, 20, 30, and 50 ml., equivalent to 0.5, 1.0, 2.0, 3.0, and 5.0 mg., respectively, of the insecticide. Successively extract with Skellysolve F, wash with water, evaporate the solvent, and develop the color in alcohol as described above. Take all readings a t 540 mp exactly 25 minutes after adding the sodium sulfide reagent. The Klett-Summerson colorimeter is equipped with a logarithmic scale. Draw the standard curve by plotting concentration in milligrams of insecticide against the dial reading. For instruments not so equipped, plot the concentration against the log of the reciprocal of the transmittance. Beer’s law is followed in the range of 0.5 to 10.0 mg. of pyrethrins, allethrin, and furethrin. Calculation. Calculate or obtain directly from the standard
Table 111. Data for Standard Curve and Experimental Error Insecticide, hlg.
bllethrin 0.5 1.0 1.5 2.0 2.5 3.0 10.0
Furethrin 0.5 1.0 2.0 5.0
10.0
Instrument Readings
Readings per Mg.
18.0 38.0 58.0 71.0 90.0 110.0 361
36.0 38.0 38.7 36.8 36.0 36.7 36.1
16.0 35.0 69.0
32.0 35.0 34.5 32.0 36.0
160 360
2
36.9
33.9
DISCUSSION
Deep red
Chrysanthemic acid, ester of 4-hydroxy-2-(p-methoxyhenzyl)3-methyl-2-cyclopentene- 1-one Dark orange vermillion n-Propyl isomer Light yellon-
Table 11.
curve the milligrams of insecticide per 0.5 sq. foot of treated paper. Subtract the correction blank in terms of milligrams per 0.5 sq. foot of untreated paper and report the final results as corrected milligrams of insecticide per square foot of paper.
Error,
%
2.9
5.4
The color reactions of sodium sulfide with allethrin-related compounds ( 2 ) , fatty materials, and pyrethrum synergists are in Table I. Compounds which developed no color with sodium sulfide are in Table 11. The data for the standard curves of pyrethrins, allethrin, and furethrin are shown in Table 111. The average experimental error of the method is about 4% and was calculated by the following formula: 82 = nZX2 - ( X X ) 2 n(n -
I)
where S = standard deviation, n = number of samples, X = value of each sample. Then the percentage experimental error is equal to:
where 2 = arithmetic mean of the value of the samples. Since continuous heating of acetone solutions of the three insecticides might cause loss through thermal decomposition, three series of tests were run using aliquots of a standard solution of pyrethrins. One series was analyzed directly without heating. The second and third series were refluxed for 3 and 6 hours, respectively. Analysis showed no quantitative differences in the color developed in any of the series. In preparation of the sample for color development, the solution containing the insecticide is extracted with Skellysolve F in order to separate the active insecticide from insecticidally inert compounds which are not soluble in Skellysolve F. The effects of this extraction are indicated by the following readings.
Pyrethrins
Instrument Readings per ?&. Extraction No extraction 19 2 35 1 19 4 35 5
Allethrin (techrucal)
37 4
54 3
Furethrin
33 9
56 4
No color is obtained if the solution is acidic when the sodium sulfide reagent is added. In the evaporation to dryness of the Skellysolve F extract, a stream of nitrogen gas may be utilized. However, air and carbon dioxide must not be used, because difficulties in the development of the color will result. The possibility of a variation in the results of pyrethrins analysis caused by a variation in ratios of pyrethrin I to pyrethrin I1 was investigated. The analyses of commercial preparations made by the Bureau of Entomology and Plant Quarantine, Beltsville, Md., show a variation in the ratio of pyrethrin I to pyrethrin I1 ranging from 1.00 to 1.32. Sample A, representing the maximum ratio of 1.32 to 1, and Sample B, representing the minimum ratio of 1.00 to 1, were selected for investigation to determine whether a variation in the results was introduced by a variation of these ratios. The effect of different ratios of pyrethrin I to pyrethrin I1 (three replications of each sample) was as follows. Sample A 18.0 19.1 20.6 Av. 1 9 . 2
Instrument Readings per JIB. Sample B 18.0 20.1 20.7 19.6
There was no significant difference between the absorbancy of Sample A and Sample B. Therefore, it may be stated that within the conditions of this method and within the limits of con-
V O L U M E 2 5 , NO. 9, S E P T E M B E R 1 9 5 3 Table IV. Insecticide Pyrethrins
1369
Quantitative Recovery of Pyrethrins, Allethrin, and Furethrin from Kraft Paper Added, M g . 5.2
Allethrin
13.9
Furethrin
5.1
Recovered, Mg. 5.4 5.0 5.7
Recovery, c/o 104 96 109
14.7
105 95
13.2
;:;
90 94 99
bv. recovery
amount of insecticide recovered. The acetone method seems to be more efficient because it gives a smaller correction blank for untreated paper and yet extracts the insecticide quantitatively. The applicability of this method as an assay for concentrates of allethrin was tested with three samples of technical allethrin. The purity, or percentage concentration, of these three samples was determined by analysis as described in this paper, the absorbance per milligram being compared with that of a sample of cydl-trans-allethrin (crystalline). Table V shows the percentage of allethrin in the three samples according to four methods of assay. ACKNOWLEDGMEKT
Table V.
Determination of Allethrin in Technical Material
Method Hydrogenolysis (alkali activation) Hydrogenolysis (correcting for anhydride) Ethylenediamine Colorimetric
Sample 1
Sample 2
Sample 3
72.5
94.1
88.9
66.3
91.7 89.8
89.8 85.3 84.1
73.0
66.0
90.0
centrations studied, there is no variation introduced in the analysis of pyrethrins by varying the ratios of pyrethrin I to pyrethrin
11. The quantitative recovery of the three insecticides from kraft paper by the extraction method described herein is given in Table IV. Pyrethrins were present as a commercial coating; allethrin and furethrin were added to the kraft paper in an acetone solution, and the paper was allowed to dry. Extraction of the insecticide from coated paper with ethyl alcohol, according to the method described by Edwards and Cueto (5),gives slightly higher uncorrected results. However, after a correction is made for the untreated paper blank, there are no significant differences between the two methods of extraction in the
The authors are indebted to Hamilton Laudani and S. 4 . Hall for many helpful suggestions, and to ill. S. Konecky and D. J. Glover for hydroqenolysis determinations of allethrin. All are members of the Bureau of Entomology and Plant Quarantine. REFERENCES (1) Assoc. Offic. Agr. Chemists, “Official Methods of Analysis,” 7th ed., pp. 72-3, 1950.
(2) Chen, Y. L. and Barthel, W. F., J . Am. Chem. SOC.,in press. (3) Edwards, F. I., and Cueto, Cipriano, ANAL.CHEM..24, 1357-9 (1952). (4) Feinstein, Louis, Science, 115, 245-6 (1952). (5) Hagsett, S. N., Kacy, H. W., and Johnson, J. B., unpublished work. (6) Konecky, XI., Schechter, LI. S., Storherr, R. W., Green, K.,and La Forge, F. B., unpublished work. (7) JIatsui, Masanao, LaForge, F. B., Green, X., and Schechter, 31. S., J . Am. Chem. Soc., 74, 2181-2 (1952). (8) Oiwa, T., Inove, Y., Veda, J., and Ohno, LI.,Botyu-Kagaku. 17, 106-22 (1952). (9) Schechter,’M. S., La Forge, F. B., Zimmerli, A., and Thomas, J . hl., J . Am. Chem. SOC.73, 3541 (1951). (10) Thompson, R. B., Chenicek, J. A., and Syman, Ted, I n d . E ~ Q . Chem., 44, 1659-62 (1962). RECEIVED for review March 28, 1953.
Accepted June 25, 1953.
Small Amounts of Uranium in the Presence of Iron Colorimetric Deter rninatio n with 8-Quinolinol LOUIS SILVERMAN, LAV.4D-4 MOUDY, AND DOROTHY W. IIAWLEY Atomic Energy Research Department, North American Aviation, Inc., Downey, Calif, The direct colorimetric determination of uranium, in the presence of moderate or large amounts of iron, i s not possible by other methods. By the 8-quinolinol method, uranium may be determined in the presence of 500 mg. of iron, 50 mg. of copper, 20 mg. of nickel, or 20 mg. of cobalt without prior separations. In conjunction with the hydrolytic separation by which uranium is separated from most of the colored ions but not iron, uranium may he determined colorimetrically, as a routine technique. With an ordinary Beckman spectrophotometer 107 of uranium are easily determined.
M
ANY procedures have been suggested for the colorimetric determination of small amounts of uranium in the presence of foreign elements. Rodden (IS) extensively outlined the peroxide methods showing their applicabilities and shortcomings and noted the effects of interfering elements, such as iron, chromium, and excess reagents. The ferrocyanide method (9,11, IS, 16, 17) also has been investigated. The objection to this method is that uranyl ferrocyanide is colloidal, and such a dispersed precipitate must be kept in a suspended state; furthermore, the ferrocyanide ion reacts with many other ions. The thiocyanate method is currently of interest ( 8 , S, IS). I n the visible spectrum the necessary reagents do not interfere
greatly; but in the ultraviolet range important interferences are noted (2, S,6). Rodden (1s) and Ware (18) offered a list of organic reagents which form colored solutions or suspensions, and from these reports the reagent 8-quinolinol was chosen for the present investigation. Greenspan ( 4 )determined uranium as the colored uranyl 8-quinolinolate in alcoholic solution. Hubbard ( 7 ) separated the insoluble uranyl quinolinolate [U02(C9H60N)2.CpH,0N 1, then coupled the 8-quinolinol to diazotized sulfanilic acid. However, Smales and Wilson (16) obtained the yellow to brown uranium quinolinolate in chloroform solution in order to determine uranium. This last technique is satisfactory for pure uranium
