Estimation of Micro Quantities of Pyrethroids ALBERT A. SCHREIBER
and
DONALD B. M C C L E L L A N
McLaughlin Gormley King Co., Minneapolis, M i n n .
attempts to estimate small quantities of pyrethiins I in flour which had been stored in pyrethrum-treated cotton
test tubes to avoid a nephelometric side effect, n dilution of the reagent avoided precipitations and hnd the additional advantage of stabilizing the reagent so that the solutions could be kept for extended periods without decomposition. Exposure t o strong light and air should be avoided, howevrr, as in the case of normal Denighs reagent. Owing to the well-known difficulties of obtaining pure pyrethrins, the method was tested anti developed with pure, synthetic chrj santhemic acid (a racemic mixture of dl-cis- and &transehrysniithemic acids boiling a t 122' to 124" C. a t 5 mm. of mercury) and r%-ithcrystalline allethrin (13). The color reaction of the latter tended to be a salmon red shade, turning into a i:ispberry led, in contrast with the more definite range from pink to a stable purple obtained with the chrysanthemic acid. The same salmon shade could be produced by adding allethrolone to the reaction of chrysanthemic acid and the reagent. The apparent shifting in color values caused by cyclopentrnolones could not be completely eliminated with the available colorimeter filters. This does not preclude the possibility of having the allethrin ieact directly with the reagent, and rather consistent results were obtained in spite of the variation in shade. However, interfering extractives, such :is cyclopentenolone and other fatty acid esters, can be Iargelv removed t)y the saponification, barium chloride treatment, and filtiation of the AOiiC method. Accordingly, these steps of the .iOAC 1950 method should be utilized in the estimation of pyrethrins I and allethrin, a t least in heterogeneous mixtures containing the latter, to provide greater over-all accuracy. Since pyrethrins I gave definitely higher results than equivalent amounts of allethrin or synthetic chrysanthemic acid, nntural chrvsanthemic acid consisting mainly of the trans isomer, wa8 isolated in 9Tr; purity and tested. Though no difference in shade iws found in the reaction with the reagent, all values obtained were a!)out 1 3 5 higher t h w those obtained with equal amounts of synthetic acid. [This difference is higher than that found by Schechter (12) in the rctluction of normal Denigbs reagent by (114s- and dl-tmns-chrys:inthemic acids. ] A\rcordingly, separate charts h w e to be prepwed for the standwdization of the different wids or materials containing thrm. The results thus obtained allonctl a s:LtisttLctory standardization. Errors found did not exceed the usual range of about 3% occurring with Klett-Summerson photoelectric colorimeters. The method was :tpplied first to commercial solutions of pyrethrins and allethrin of rather low concentrations, intended for purposes in which the above degree of error was considered immaterial. Thereafter, formulations containing these pyrethroids in combination with DDT, synergist MGK 264 (98% ,~-2-ethylhexyll-,ir?.clo[2.2.1]-~-hrptene-2,3-dicarhoxiniidc), piperonyl butoxide (80Tc a-[2-(2-butoxyethoxy)ethoxy]-4,5methylenetlio\y-2-prop? ltolucnr : ~ n d20'5 related compounds), or I d h a n e 384 ( 5 0 5 8-hutosy 8-thiocyano diethyl ethcsi ), oleic acid, rancid corn oil, or severd of these materials were tested, and they gave sufficiently accurate result? for an estimation of their pyrethroid content. -4s a major problem, encountered in connection with previous work ( l q ) , flour from pyrethrum- or allethrin-treated bags and such cotton and paper bags themselves n-ere tested. (In the case of niateriala containing p) rethrins i t should be remembered that the piesent method applies only to pyrethrins I and the ratio of these to pyrethrins I1 m:iy vary from 0.8 to 1.5 depending on the origin of the pyrethrum flon-ers used and possihlr changes in the final extracts therefrom,) Although the extrxtion of
\ RECEYT
bags ( I $), the extraction from cotton, paper, and other media wap found to be difficult and there were apparent limitations in the accui:wy of the analysis of pyrethroids-i.e , chr\ s inthemum esters ot alkyl cyclopentenolones by the Associntion of Official hg?.icultural Chemists (AO..lC) 1950 Method (1). Since then, these problems have been given further consichit ion in this 1at)or:ttory I t has heen suggested ( 4 ) that the sulfuric acid used in the piecipitation of excess barium chloiide in thc .ZOAC 1930 method be replaced by hydrochloric wid. This modification would avoitl the b:trium sulfate precipitate altogether and, there!)y, the pnssibility of adsorption or occlusion of chi J sdnthemum acids. On testing a substantial number of baiium precipitates of routinr analyses in this laboratory, it was found that such adsorption can be easily discovered in the precipitate or on the filtei disk bv the color reaction with Denigee reagent. However, :idsorption could be produced only by abnormal procedure, such as filtintion of more than one batch of precipitate on the same filter disk, the u v of a Ruchner funnel with a t least pzrtly clogged pores, or insufficicnt washing which mny he caused by variations in suction. en then the quantities :ibsorbed amounted only to frnctions ot those found by Mitrhrll(11) upon extraction of barium precipitates with warm, aqueous 1.V sodium hydroxide Any letention !)eyond the limits of error of the method does not t:ike place when the analysis is carried out as directed. I n the courqe of these e\periments it was found th:it :L few niicrograms of pure, synt tietic chrysanthemic (chrv~anthemummonocarl)o\vlicacid) ncid spread on the filter d i d fiom a solution in petroleum ether, ieadily produce the whole gamut of colors of this reaction. The color strength suggested th:it thc ad3orptions were of the order of about l / l o o of ~ the amounts of p ~ r e t h r i n sI and of allethrin normally used in their determination, such as from 70 to 100 mg Accordingly, a conversion of the AOAC method into a related rnicromethod seemed possible. I n any case the formation of :E barium precipitate proved :dv:mtageous (compare 17') for rrtnining extraneous matter of fatty acid nature as well :is oily polymerization products such as ciin hr formed in the saponification of cyclopentenolone esters (6, 9). Thesr, like allethrolone itsrlf, CYII reduce DenigEs rwgerit with the formition of :L vellowish t ingc. As previous work had shon-n ( 1 4 ) that Denigbs reagent can be reduced by materials other than chrj santhemic acid without the formation of a colored precipitate, i t was considered that the :ipparently specific color reaction of Denigbs reagent with chrysznthemic acid might give values uninfluenced to a substantial degree by extraneous niatciial. I t was also hoped to avoitl eirors likely to occur with pyrethrins in the AO-IC 1947 Method :Lilt1 errors in spectrophotometric analysis in thc u1tr:ivinlet i.inge ( 3 , 16) caused by the presence of biologicdly inactivr nisitciial, such as poll merizcd material and unsaturatrtl fnttv xcid caters (8). The latter may overlap the curves of the pvrethroitls h colorimetric determination of chrysanthemic acid has been suggested by Fischer ( 5 ) , a stabilization of the color occurring Kith Denighs reagent being attempted by a substantial increase i n the concentration of sulfuric acid over that suggested bl- Autliftien ( 2 ) . I n spite of the suggested filtration of the highly acid I rngent, further precipitations have often been encountered in the reaction of the acid with the reagent Although these precipitntrs cnn he centrifuged to the bottom of the colorimeter 604
V O L U M E 2 6 , NO. 3, M A R C H 1 9 5 4
605
pyrethrins and of allethrin from flour and of pyrethrins from paper or cotton bags did not present any particular problem in most cases, the extraction of allethrin from these wrapping materials presented problems which could be explained and at' least partly solvctl only by a consideration of the physical problems of adsorption as encountered in chromotography. At the present time the nature of these problems, apparently based mainly on irrrversible adsorption, has been recognized but the difficulties have not been completely overcome, as, for example, in the :ittempts at a chromatographic separation of pyrethrins and of their degradation and reaction products (10, 19). Extractions based on experience in adsorption and elution (16, 18) were rather successful. I n those specific cases in which n p:irtinl or complete irreversible adsorption may have talien place, caused by the choice of solvents in the application of allethrin or by adsorbing hintleis or sizes in fabrics and paper, thc Denighs reagent may / ) e :Ipplied directly to the impregnatd materials and the color thus Eornictl compared with a standwd prepared from measured amounts of pyrethroids applied to the untreated material. S:~turally,such procedure can give only rough approximations. I n any case, the present method avoids thr necessity of using the inconveniently large samples of suhstratc heretofore required i n thc estimation of smnll qumtitics of t h c pyrethroids ( 7 ) . REAGENT
The reagent of Fischer (.5) differs from AOXC ( 1 ) i n that thr reaction mixture of mercuric oxide, sulfuric acid, antl w t e r is diluted with 5 volumes of w t r r , filtered, and then 1.5 volumes of sulfuric acid are added to the cooled filtrate. Though a strong pink color turning into purple c:m be obtained on adding sodium chrysnnthemate to the reagent,, almost invariably a precipitate is formed Rhich may obscure the reading in the colorimcter. Centrifuging for about 45 seconds n t about 1200 r.p.m. settled thc precipitate a t the bottom of the colorimeter tube, but it is found that readings from varying quantities of chrysnnthcmic acid down to about 10y wcrc consistent onlj- with each specific hatch of reagent. I n addition, it proved practically impossible t o prepare several reagent mixtures giving identical reatlings wit,h eqLial quantities of chrysanthcunic wid.
Table 1. Specific Calibration Values Obtained f r o m Natural and Syntheticn Chrysanthemic Acids Pynthrtic Acida, y Reagent blank 5 10 20 An 60 80 100 120 140
Coloriineter Sde Re:\ilinl:
h-aturcil Acid, y
23 32
31
__
160 180 m - _n_
100 &cis (from synthrtic acid) 100 dl-trans
(from synthetic acid)
Q
40 5-I ..
86 118 148 179 212
52 85 104
13.5 165
Colorinietcr Scale Reading
77 117 176 208 264 321
'740
:72 300 3.10
on 210
('oiiimrrcial. racemir nciil.
However, it was found that :L satisfactory- reagent could I)(, prepared by a 10-fold incrensr of the quantities of water and ol sulfuric acid in the AO.iC DenigPs reagent-that is, by using 40 ml. of sulfuric acid nncl 80 ml. of water per gram of yello\T mercuric oxide. This reagent did not produce n precipitate, furni.hed a stable color reaction a t the maximum of absorption, protlucwl consiptent results, Tntl hnd a good shelf-life when pro-
tected from excessive light, air, and high temperatures. portion of the reagent that had stood for two weeks in subdued light at temperatures from 25' to 30' C. gave accurate readings when retested with known samples. Also, different batches of reagent msde with the samc concentrations produced identical results. Cniform readings following Beer's h r v were obtained with the Klett-Summerson colorimeter a t 25" to 30" C. for s'imples of from 5 to 2007 of synthetic chrysanthemic :icid and up to about lOO+f of nntur:il chr>s:mthemic acid (Table I). A variation in the acidity of the reagent will vary thc intensity of the color. If greater sensitivity a i t h smaller s.imples is tlesirrd, 1 educiiig the concentration of mercuric oxide :md incrcasing that of sulfuric acid will produce a more intense coloi. For example, 5 ml. of reagent prepared as described and reacted with 1007 of synthetic chrysnnthemic acid gaive a re:tding on the cwlorimetcr s c d e of 180". A reagent with 3OC, less mercuric o\ide and loc; more sulfuric acid reacted with lOOy of thr chi) s:intheink acid lint1 gave a reading of 242". PROCEDURES
Preparation of Reagent. Mix 1 gram of yellon nieicuric oude s i t h 80 nil. of water. With stirring in a cold water bath, slowly add 40 nil. of concentrated sulfuric acid (at Iwit 9 5 5 ) nnd stir until completely dissolved. Preparation of Standard Reference Curve. Keigh accurately about 100 mg. of pure, synthetic chrysantheiiiuiiimonoc:Lrbo.;~lic. acid into a 100-nil. volumetric flask and dilute to volume ~ i t h lowboiling petroleum ether (boiling point, 20 to 40' C.). Dilute a sufficient portion of this solution with petroleum ether to produce a solution containing lOOr of the chrysanthemic arid per milliliter. Pipet aliquots of this final dilution directly into a 10-ml. tube of a Klett-Summerson photoelectric colorimeter (or similar instrument). Evaporate the petroleum ethw by holding the tube at an angle over a small opening of a boiling water bath while agitating, taking care not to remove the labt trace, as overheating may drive off some chrysanthemic acid. Immediately cool the tube to about 25 O C. by immersion in cool water. Rapidly pipet 5 ml. of the reagent into thc tubr, it shake to ensuye uniform nds to remove air bubbles. niiuing, and centrifuge for 30 Place the tube in Klett-Su colorimeter with S o . 54 filter, read a t about 1-minute intervalIlite, diatomaceous earth, from solutions in low boiling petroreum ether the estraction can be carried out as described in the testing for contamination of flour, subsequently taking a n aliquot representing about lOOr of pyrethrins or allethrin. Otherwise, the extraction and concentration are better carried out as described for estimation of allethrins or pyrethrins I in paper or cloth bags. I n each case the further procedure is the same as in the tests on flour. DISCUSSION
S o difficulties were encountered in applJ-ing the present method to solutions of the pyrethroids or to adsorptions thereof on flour and on neutral, inorganic extenders or in a size prepared therefrom for impregnating muslin. Hov,*ever, interesting phenomena of at least partial irreversible adsorption and consequent incomplete extraction were encountered with adsorptions on paper _ _ and on frit'ted- glass, especially byhen petroleum ether was used as the solvent in the adsorption. Table IT. Effect of Substrate and of Solvents on Recovery of Pyrethroids I n most of those cases in which loa results were found, ColorImpregnated imeter a treat,ment of the extracted with Scale NO. Substrate All.a Chry6.b Solvent Extracted wit11 Reading Reco\-ery, 0 substrate with the reagent and 1 10 Grams wheat flour 8050 P.e.C P.e 102 102 the formation of color indi2 180 100 178 98 cated very clearly an incom3 270 150 262 102 4 360 200 330 99 plete estraction even before the 5 2.6 Sq. in. Whatman 180 100 Bcctone 141 76 so. 4 analysis of the extract was 6 2.1 Sq. in. Whatman 144 80 95% Ethanol 0 0 completed (Table 11, ?\'os. No. 4 7 144 80 95% Ethanol plus 75 41 6-14, 17, IS,. The adsorps KaOH 61 30 9 144 80 95% Ethanol plus tions prepared from solutions 0 0 0.1% HC1 in petroleum ether were sub10 I44 80 Dioxane plus 2 % HzO 91 53 61 30 stantially more "resistant" than 180 100 Washed p.e. 100 49 those prepared from solutions 114 80 Stoddard F plus 1 5 % 18 20 containing some more hyclromethanol 111 80 Stoddard F plus 10% 66 34 phylic or higher boiling solbenzol 144 80 Deodorized kero- 95% Ethanol vent, such as deodorized kero127 84 sine + 4 vol. sine (Table 11, No. 15, 16) as p.e. 16 I44 80 95% Ethanol plus 135 90 is usually present in ext,racts KaOH 17 I44 80 P.e., dried, niois- 95% Ethanol (neutralof pyrethrum flowers. 74 40 tened with ized) I n such cases, which are not kerosine 18 144 80 95W Ethanol 60 30 unusual ( 1 7 ) , the est,rsction 19 144 80 Saponified, then 108 67 p.e. was forced with different, higher 144 80 20 P.e. Dioxane, 1% acetic 113 73 boiling solvents such as diacid 21 2.6 Sq. in. Whatman 180 100 Dioxane, 1 % acetic 183 101 oxane and butanol and by No. 4 acid, 2 7 , Hz0 22 180 100 the addition of soluble acid Dioxane, 0.270 Santo162 90 23 144 80 merse, 4% HzO 125 81 and surface active agents such 180 100 Dioxane, 0.2% D.B.d, 180 100 144 80 4% HzO 148 100 as acetic and dodecyl ben144 80 146 99.4 zenesulfonic acids and Santo144 80 ethanol, 0.2% D.B.d, 98 60 merse 3. The estractions of 2% HzO 28 234 130 hcetone Acetone 200 87 dioxane and butanol were not 29 234 130 95% Ethanol 200 87 30 2 6 Sq. in. sized muslin 144 carried out in Soshlet estrac80 P.e Dioxane, 0.2% D.B.d, lose 65e 4% Hz0 tors but, by simple warming 31 180 100 182 101 32 180 100 100e 49c in Erlenmeyer flasks either re33 180 100 peatedly with small quantit,ies 182 101 34 180 100 176 98 of solvent or just once with 35 180 100 178 99 ext'ended viashing. T o avoid disappointing ex36 180 100 73 127 periences in using the above 5 Sq. in. kraft paperf 37 Pyrethrins 446 12.22 31g.pyi. I w r . I/method for the estimation of per sq. ft. pyr. 11= pyrethroids impregnated in 1.2 37a 2.6 Sq. in. kraft paper Allethrin, 109 11.9 Afg per sq. commercial packing materials, approx. 10 mg./sq. ft. technical it. 38 2.1 Sq. in. kraft paper, Blank it should be noted that, in untreated 39 5 Grams pyrethrum some cases, it was not possiP.e. 76 101 (Pyr. I) dust, 0.23 pyrethrins ble to obtain a satisfactory (pyr. I/pyr. I1 = 0.9) % w. extraction in contrast to esa Crystalline allethrin. d Dodecyl benzenesulfonic acid. tractions of impregnations preb Synthetic chrysanthemic acid. e Distillation of dioxane carried too far. Petroleum ether, boiling point 20' to 40' C. f Experimental commercial impregnation. pared in this laboratory with similar substrat,eseither shortly ~
C
607
V O L U M E 26, N O . 3, M A R C H 1 9 5 4
(8) LaForge, F. B., and Acme, F.. Jr.. J . Ora. Chem.. 2, 308 (1937). (9) LaForee. F. B..Green. N.. and Sehechter. At. S.. J. A n . Chem. Soc.~74,5392(1952): Lord, K. A,, Ward. J., Cornelius, J. A,, and Jilrvis, PI. W.. J . Sci. Food AB?., 3, 419 (1952). Mitchell, Wm., Ibid.. 4,246 (1953). Schechter, M, S., private communication, 1951. Soheehter. M. S..LaForge. F. B.. Zimmerli. A,. and Thomas. J. M., J . Am. Chem. Soc.. 73,3541 (1951). Schreiber, A. A,, and McClellnn. D. B.,ANAL. CHEM.,24, 1194 (1952). Shukis, A. J., Christi, D., and Waehs, H., Pyrethium Post, 3, 20 (1952). Strain, H.H., "Chromatographic -4dsorption Analysis." Vol. 11, p. 66,New Yolk. Interscience Publishers, 1940. Takei, S., Omo. M., and Nakasime, M., J . Agr. Ch.em. Sac. Japan,16,389-410 (1940). Trappe. H., Biochem. Z . , 305, 150. 306. 316, (1940). Winteringham, F. .'l W..Science, 116, 452 (1952
after their preparation or after extended storage. Apparently, some binding, siaing, and coating additives used in the production of the packaging materials can exert an even larger adsorptive power than most of the substrates used in the present investigation. On the other hand, the present method permits qualitative identification of residual traces of the pyrethroids or chrysanthomic acids in their production and use. LITERATURE CITED
(1) Assoo. Offio.Agr. Chemists, "Nethods of Analysis." 7th ed., P. 72, Washington, 1950. ( 2 ) Audiflren, M.. J . p h a n . chin., 19, 535 (1934). (3) Beekley, V. A,, Purethrum Post, 2, 23 (1950). (4) Beokley, V. A,, and Hopkins. J.. private communication; J. Assoc. Ofic.Agr. Chemists, 35, 64 (1952): 36, 66 (1953). (5) Fiseher, W.,Z . anal. Chem., 113, 1 (1938). (6) Harper, 5.A,, Pwethrun Post, 1, 15 (1949). (7) Jones, H.A,, Kerbey, G. F.. and Incho. E. J., Chem. Special&$ Mfrs. Assoc., P ~ c .38th . M i h Y r . Meeting 1969. p. 94.
Reoerveo for review July 16, 1953. AeoeDted Deoernber 1
Determination of Sulfur and Barium in Organic Barium Sullfonates F. E. BRAUNS, J. B. HLAVA, and HEINZ SEILER The institute of Paper Chemistry, Appleton, Wis. HE quantitative determination of barium and sulfur in organic r r barium sulfonates involves certain difficulties. I n water-
soluble sulfouates barium may be determined by precipitating it with sulfuric acid and determining it 8 8 barium sulfate; in water-insoluble sulfates it nisy be estimated by ashing the compound and determining the barium in the sulfated ash-if no other ash-forming elements are present. The latter method may a180 be applied t o barium lignosulfonates, since i t is impossible to precipitate the barium in these salts with sulfuric acid because the barium sulfate remains partly in colloidal solution and cannot be filt.ered quantitatively as a result a i the peptizing effect of the lignosulfonic acid. The determination of sulfur in organic barium sulfonates is more difficult. The ratio of barium to sulfur in normal organic barium sulfonates is 1 t o 2; in other words, there is twice as much sulfur present as can be consumed by the barium in the formation of barium sulfate. I n a Carius or a Parr bomb determination, half the sulfur is converted into barium sulfate and the other half int.o sulfuric acid. The barium sulfate which is formed would have to he filtered quantitatively, the sulfuric acid in the filtrate would have t o be precipitated with barium chloride, and theresulting barium sulfate would then be filtered and weighed. I n order to detect relatively small differences in the degree of sulfonation of lignosulfonic acids, which are often isolated as their barium sdtlts, i t was desirable to have a method available which would permit the determination of both barium and sulfur in such barium lignosulfonates and, if possible, in the same sample. To reach this goal i t was necessary to separate the barium quantitatively from the sulfur in such a way that each could be determined. Attempts to separate them by subjecting a weighed sample of barium lignosulfonate to an oxidizing fusion with an equivalent mixture of sodium carbonate and sodium nitrate (whereby the barium was obtained as insoluble barium carbonate and the sulfur as sodium sulfate) appeared promising but required special precautions and was too time-consuming. A much simpler quantitative separation of barium and sulfur can be achieved by the use of a 8mdl cation exchange column. The barium is zdsorbed on the exchttnge resin, whereas the sulfonic acid is obtained quantitatively in the filtrate in which the sulfur is then determined by the method of Salvesen and Hogan (1). The quantitative elution of the barium from the ion exchange column was a t first unsuccessful because the acid usually used
in fact, almost no barium could b r eluted with 10% hydrochloria acid. On elution with 20 to 25% hydrochloric acid the barium could be estracted, but t,he process was slow and the barium values wereoften too low because the amount or hydrochloric acid apparently was insufficient. The principal fault seemed to be t h a t the barium had to pass through the whole column and was partly readsorbed by the resin until the hydrochloric acid again became strong enough locally to keep the barium in solution. The use of a still stronger acid caused considerable swelling of the resin; this swelling may be great enough to cause the absorption tube to burst. To avoid passage of barium through entire COIL umn, a new tube with standard ground glass joints a t each -"A nmo rl.*iono ..uw.a..-d allowing barium to be eluted from the other end of the column thus preventing its passing through whole ion exchange resin. Figure 1. Ion Exchange Colnmn
For the separation of barium irom sul-
