(375) Venkateswarlu, Ch. Nallett, 11. W. ~ N A L .CHEM.32, 1888-90 (1960). ( 1 i 6 ) ' i'inogradova, E. N., Vasil'eva, IJ. N.J Iobst, K., Zavodsk. Lab. 27,
525-7 (1961).
(177) Volodarskaya, R. s., 1bid.j 26, 925-7 (1960). (178) Wscha, E,, JIetall 14, 906-7 (1960). (179) Walkden, J., Heathfield, K. E., At. Energy Research Estab. (Gt. Brit.) AM 32, 1-3 (1959).
(180) Webb, 11. S. W., Shalgosky, 11. I., IC. I., Fiz. Sbornik. L'cou. L'nib. 1958, C . K. At. Energy Authority, Prod. 206-7. Group, PG Rept. 171, 119-27 (196Oj. (184) Zimmer, IC., Torok, T., .Ida (181) Willard, H. H., Dean, J. A,, - 4 s ~ ~ . Chim. Acad. Sci. Hung. 24, 1-17 (1960). CHEX.22, 1264 (1950). (185) Zimmer, K., Torok, T., A n n . (182) Zaidel, A, N,, ~ ~ l i t ~ ~s, ~ I,, ~ k i i ,Univ. Sci. Budapest. Roland0 Eotvos Lipovskiy, A. Razumovskii, 1.) A\'oniinatae, Sect. Chim. 2 , 383-8 (1960). Ynkimova, P. P., Fiz. Sbornik L ' i ~ o c . (186) I b i d . , 2 , 395-402 (1960). T;niu. 1958, 3T-10. (187) Zirichenko, \r. -I.,Iiudina, S. I., (133: %:lidel.-1.I., Pr.trov, A . .I.,I'etrov, Zauodsk. /AD. 27, 956-3 (1961j .
Pesticides William E. Westlake Agricultural Research Service, Entomology Research Division, U.
period from Sovember 1960 through October 1962, covered by this review, has been charact'erized by striking advances in techniques for the microdetermination of pesticides. The most outst,anding developments have been in instrumental techniques, some of which n-ere mentioned in the last review of this series by Westlake (88), and discussed by Schechter and Westlake (78). Refinements and modificatiom of the gas chromatograph have made possible the routine screening of samples for many of the commonly used pesticides at levels far below those that c,ould be detected by routine methods two years ago. Improvements in infrared equipment and techniques have made possible the quantitative determination and qualitative identification of pesticides with this instrument in the micrograin range. Thin-layer chromatography, still in the early stages of development, promises to be a valuable tool for determining pestkide residues. The high capacity of the thin-layer plates, in comparison n-it11 paper, permits the use of large samples and the isolation of amounts of the compounds under study large enough to allow their identification by infrnred or other means. Seut'ron activation, not a new technique, is just beginning t o be recognized as having great potent'ial for determining residues of pesticides that contain an clement that can be activated by exposure to it neutron flux. This method is of particular interest when a large number of samples are to be analyzed for one compound, because of the simplicity of sample preparation, speed, and low cost per sample. This review deals exclusively with methods for determining residues. hlethods ior analyzing formulations and technical products are available through t'he hlethods Clearinghouse of the Asociatioii of American Pesticide Control Officials. HE
S. Department of Agriculture,
GENERAL METHODS
Gas Chromatography.
T h e adaptation of gas chromatography to the determination of pesticide residues ha5 received more attention during the past two years t h a n a n y other one method of analysis. Because such investigations have almost all been initiated during this period, the number of publications concerned with gas chromatography of pesticide residue\ is not large. The situation nil1 undoubtedly be different during the next two years. The impact of recent developments in gas chromatography has been great, and was discussed hSchechter and Westlake (78). l'ht ability of the newly developed type< of detectors, particularly the electronaffinity detector developed by Lorelock (&?), to detect exceedingly small amounts of organic compounds, has had a twofold effect. First, i t is noIy possible to determine, quickly and accurately, levels of pesticides that could not previously have been meaured. Secondly, when the ultimate 111 sensitivity is not required, the iize of the sample subjected to analysis can be reduced to such an extent that the difficult and time-consuming cleanup procedures aqsociated n ith many methods used heretofore can be eliminated. Burclifield and Storrs ( 8 ) have included a section on pesticide residue.. in their excellent book on biological applications of gas chromatography. Practical applications of gas chromatography are stressed, but enough tlieoretical background is given to meet thc requirements of the average analyst. Goodm-in, Goulden, and Reynolds ( S I ) have developed a rapid procedure for identification and quantitatiw determination of residues of chlorinated hydrocarbon pesticides in crops. I n their rapid sorting test they used a 2foot column packed Ivith 2.5% of E301 silicone elastomer and 0.25% of Epikote 1001 on 100- to 120-mesh ltiedguhr.
Beltsville, Md.
Lindane, heptachlor, aldrin, Telodrin (1,3,4,5,6,7,8,8-octachloro-l ,3,3aJ4,7,iahexahydro - 4 , i - methanoisobenzofuran) , dieldrin, endrin, and DDT could be identified in crop extracts. All except DDT were determined a t levels of 0.1 to 0.25 p.p.m., and DDT a t 1.0 p.p.m. Chlordane, toxaphene, and methoxychlor could be detected at higher levels. The ordinary argonionization detector, far less sensitive than the electron-affinity type, was used in this work. Wells (87) has reported a rapid -,weening procedure for many of the commonly used insecticides, including nine organophosphorus compounds. The electron-affinity detector was used, and levels of 0.02 p.p.m. or less were readily determined in 20-gram samples of fruits and vegetables. Samples were extracted with acetonitrile, diluted with water, and partitioned into petroleum ct'lier. No further cleanup was required. The author states that 1 p.p.b. (part per billion) or less could be detected if an efficient cleanup step !vas used. Cassil (11) has given a detailed desription of the microcoulometric gas chromatograph and its use. Extraction and concentration procedures are discussed. Expected percentage recoveries through the column are given for 25 perticides. Retention-time ratios are also given for a large number of peiticides. The results of analyses Ivith the gas cliromat'ograph are compared with those from chemical analyses for Tedion (2,4,4',5-tetrachlorodiphenyl sulfone) , endosulfan, and ethion, on several crops. A removable in.ert for the injection port is described as a means for eliminating difficulties rxperienced due to the catalytic decomposition of some pesticides in the injection area as a result of deposits of plant materials accumulating on the hot surfaces. This simple modification of the instrument has eliminated o i ~ eof VOL 15, NO. 5 , APRIL 1963
105 R
the more serious obstacles iii the determination of some common pesticides, including D D T . Burke and Johnson (9) tested the microcoulometric gas chromatograph on 77 pesticides, and successfully chromatographed 71 in 30 to 40 minutes under the conditions used. Of the six compounds that' did not chromatograph, five were phenoxyacetic acids. Relative retention times for the 71 compounds are given and cleanup procedures discussed. Yip (89) has reported a sensitive and reliable method for determining 2,4-D and other chlorinat'ed phenoxy alkyl acids by using this instrument. l l i e acidified extracts from the crop samples were extracted into chloroform, then esterified with diazoniethaiie to form the methyl ester. The esters chromatographed satisfactorily and as little as 0.05 p.p.m. could readily be determined. A method for determining 2,-1-D in citrus was developed by Erickson and Hield (%). The 2:4-D w s esterified with diazomethane and debermined quantitatively with a niicrocoulometric gas chromatograph. Zweig, Archer, and Raz (BO) have described a method for determining naphthalene acetic acid and its methyl ester. Residues of the two cornpounds were isolated from int,erfering plant subsbances by gas chromatography. The acid was esterified with diazomethane to permit chromatography. The fractions of eluent from the column were collected and absorbance was determined at 281 or 224 mp for quantitative measurement. Hughes and Freed (.$I) have developed a procedure for determining residues of ,~,S-di-71-propylthiolcarbamate (EPTC) in soil by gas chromatography. As little as 10 pg. could be determined, by using a thermalconductivity det'ector. The sensitivity could undoubtedly be iniproT ed by using an ionization detector. ri sample could be run in about 15 minutes, by using a 4-foot column packed 11-ith25YGApieaon L on Celite, a much more rapid procedure than previous methods. Phillips, Pollard, and Solon-ay (7.4) investigated the behavior of endrin in gas chromatography. They observed that endrin gave two peaks, neither of which was due to endrin itself, and these were identified as the knorvn pentacyclic ketone and a new isomeric aldehyde. Their formation vias due to isomerization of endrin in the column, This isomerization was rapid and essentially complete a t the temperature required for analyses (230" (2.). The resultsemphasizethe need for caution in interpreting results from high-temperature gas chromatography but do not negate the analysis of endrin residues by this method.
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ANALYTICAL CHEMISTRY
Other Instrumental Methods. Gunther, Blinn, and Carman (35) utilized the strong absorption bands a t 1017 and 959 cm.-l to determine ethion residues in lemons a n d oranges. Partitioning from n-hexane with acetonitrile, and chromatographing on a Florisil column ivere used to eliminate interfering substances extracted from the fruit. The method would measure as little as 15 pg. of ethion. Gershman (27) dereloped an infrared method for determining endrin re-idues. Cleanup wa5 accomplished with a chromat,ographic column containing a mixture of sodium sulfate, dt'tapulgus clay, Cclite 545, and carbon. The dried residue was dissolved in cnrbon disulfide and the spectrum recorded from 7 to 16 niirron;. The peak at 10.2 microns \\-as used for quantitative estimation. The method, as reported, lacks the sensitivity desired for re-idue studics, hut this shortcoming could be corrected by using microcell. anc1,'or ordinate-scale expansion. Castro and Schmitt (12) have used neutron-activation analysis to determine brominc, chlorine, manganese, sodium, and potassiuni residue5 in citrus. =1 completelj. instrumental procvdurt: was used and the five elements were determined quantitatively from the same set of gamma-ray spectra. &inn aiid Potter (84) used instrumental neutron-activat'ion anal determine bromine residues in a of crcjp3. Levels a. Ion as 1 p.p.m. were detectable in crop samples and as little as 0.01 p.p.m. could be detected in extract's of treated crops. I3aumlei. and Rippstein ( 2 ) studied the separation of some thiophosphoric esters and organo1)hosphorus insecticides by thin-la!*cr chromatograpliy. Several chlorinated hydroearhoii inwticides were also used. R,, valuea are given for the compounds st>udied, and the adsorbents. solvent sJ-stems, reagents, and (~perat~ing conditions are dewibed in tlrtail. _\lacDougall ( 5 4 ) presmted a11 excpllent discussion of the use of fluorescent Ilrocedures for pesticide rrsidue analysis. Cleanup methods, Ijarticularly imiiortant in this type of analysis. were given brief mention. fluoromet,ric methods now in II also described. The sensitii fluoromrt,ric methods! according to the author, is limited by the background. Levels of 0.01 p,p.m, may he readily measured if the background can be reduced below this lrvel. Giang (SO) has dereloped a fluorometric method for dctermiiiing Ihyer 22.405 ( 0 , O - tlict,liyl - 0 - nalihthalimide 1,hosl)horothioatr) in milk and buttrrfat. A fluorezcmt compound m s Ijroduced by rracting the w-iduc,s with niethanolic sodium hytiroxititx xnd dioxane in the I)rcvncc of hytii,ogen
peroxide. The absolute sensitivity of the method was 5 fig., or 0.05 p.p.m., in a 100-gram milk sample. Lisk (61) discussed the use of a modified Schoniger flask for determining chlorine, bromine, mercury, selenium, arsenic, manganese, and nickel. This was primarily a review of work prei,iously published elsen here. MacDonald (68)has also reviened the use of the oxjgen flask for determining various elements and compounds. Gutenmann et al. (37) have developed procedure. for determining arsenic, bromine, chlorine, manganese, and nickel in plant material, by using the oxygen flask. Analyses ivere made on potatoes, cherries, onions, cabbage, and oats. St. John and Lisk (77) have developed an improved procedure for chlorine analysis by the oxygen-flask method. Yew techniques were used for preparing and handling sample holders and an automatic cone-filling device nas used for samples lorn in chlorine. -4 further refinement was the use of a magnetic stirrer to eliminate qhaking of the combustion flask by hand. Kotakemori and Hauda (46) have used the Schoniger flask to burn samples for determining organophosphorus pesticides. The combuqtion products were absorbed in 27y0 perchloric acid and the phoqphorus n as determined by the molybdenum blue colorimetric method. The procedure m s sensitive to less than 10 pg. Bache and Lisk ( I ) have described a polarographic method for determining pentachloronitrobenzene on forage. Despite a rather drastic cleanup involving freezing and adsorption of some extractive. on Attaclay, follom-ed by chromatographing on Florid, the method is more rapid than that previously used. Amounts as low as 0.2 p.p.m., in 150-gram samples, nere recovered and determined. Oscillographic polarography has been applied to organophosphate pesticide residueg by Gajan (25). This recent development in polarographic equipment ha; proved to be a useful tool for residue atudieq. Polarographic studies were uqed in conjunction with paper chromatography to isolate and identify the compound.. Further n-ork appears justified on the 1,a.i. of thi? limited study. Ott aiid Gunthei (72) have developed a procedure for determining parathion and malathion in admixture, by u.ing the polarograph. Parathion at 3 pg. and malathion a t 9 pg. could be determined quantitatively in the same qolution. CHLORINATED HYDROCARBONS
General Methods. Mills (64) conducted a study of paper chromato-
graphic procedures for determining D D T , TDE, D D E , heptachlor epoxide, BHC, and lindane. Cleanup and extraction procedures and solvent systems are given. Mitchell (68) reported a paper chromatographic method for separating dieldrin and endrin. Solvent systems and conditions are given for acceptable separation of the two compounds. Polar and nonpolar Ucon lubricants were used as immobile solvents. Koblitsky, Adams, and Schechter (46) developed a rapid screening procedure for residues of ohlorine-containing pesticides in fat. The method, intended for residues of 5 p.p.m. and above, is not satisfactory for lower levels. A rapid cleanup of the fat, followed by treatment of t'he residue ivith sodium dispersion, and automatic coulometric titration of the inorganic chloride, \vere employed. Specific Methods. George, Fahey, and Walker (26), Hughes (40), and Eiduson (df) havd reported methods for analyzing Kelthane [l,l-bis(p-chlorophenyl)-2,2,2-trichloroethanol]residues. All three procedures are based on the Fujiwa,ra color reaction but differ in the steps preceding color formation. Eiduson's method lacks the sensitivity that is desirable whereas Hughes' procedure is time-consuming in comparison with the others. The method of choice appears to be that of George et al., because i t is very rapid, is sensitive to less than 10 pg. of Kelthane, and requires a minimum of equipment and reagents. Fahey and Schechter (23) have reported a modification of the colorimetric method for endrin analysis. Users of the method had experienced unduly high and variable reagent blanks and its use had been limited for this reason. It was found that the difficulty arose from the side reaction between excess sulfanilic acid and diazotized sulfanilic acid and that this could be prevented by adding an excess of sodium nitrite that was later destroyed with ammonium sulfamate. Gordon, Haines, and Rosenthal (32) tieveloped a method for determining reiiduea of Perthane [l,l-dichloro-2,2bi4p-ethylphenyl)etliane] in cow's milk :md fatty tissues from rats. Dehyilrohalogenation is followed by reaction with strong sulfuric acid to procliice a colored carbonium ion having maximum absorbance a t 495 mp. Extraction and cleanup procedures are given, in detail, for both kinds of sample. Gunther, Blinn, and Ott (36) have developed a rapid method for determining DDT in milk and butter. This procedure was intended for use in routine screening of milk supplies and The entire .-peed was imperative. process requires only 1.5 hours, and is srnbitive t o 0.2 p.p.m. in a 100-gram
sample of milk. The residue is separated from the fat by partitioning from n-hexane with acetonitrile, then nitrated, extracted into benzene, and reacted with isopropylamine-benzene solution. The absorbance maximum is a t 430 mp. McKinley, Savary, and Webster (58) have reported separation of mixture of T D E and p,p'-DDT by paper chromatography. The compounds were then eluted separately and determined quantitatively by the customary colorimetric method. N u r p h y and Barthel (71) improved the colorimetric method for hept'achlor and heptachlor epoxide residues t o permit determination of 0.01 p.p.m. of eit,her compound in soil. This improvement was accomplished primarily by using a column for chromatographic cleanup that lowered the background readings to near zero. Rusk and Fahey (76) identified a compound that had interfered with the colorimetric analysis of heptachlor epoxide, in ,some instances, and devised a technique for its removal. The interference, gamma-chlordane, was separated from heptachlor and heptachlor epoxide on a Florisil column, n-hen appropriate aolvrnt systems were used. ORGANOPHOSPHORUS INSECTICIDES
General Methods. Chilmell a n d Hartley (14) have presented an excellent review of methods for det'ermining 17 organophosphorus insecticides in foodstuffs. Details of extract,ion, cleanupj and analytical procedures are given. Laws and Webley (49) have developed a general procedure for determining organophosphorus compounds in vegetables, by utilizing solubility properties and column chromatography to achieve yeparation. Total phosphorus is determined by wet ashing, followed by use of the molybdenum blue colorimetric method. Caverly and Hall ( I S ) observed erratic re.sults vr-hen they used the method of Laws and Webley, because of partial extract'ion of the molybdenum blue complex into the aqueous pha.;e when the 2-butanol-benzene solution of molybdophosphoric acid ivas reduced by shaking n-ith acid stannous chloride solution. This difficulty was eliminated by using a freshly prepared 0.27, solution of stannous chloride in absolute ethanol. Getz (26) has devised a procedure for the identification of residues of six organophosphorus insecticides in kale. The procedure should be equally effective for ot'her leafy crops. The samples were extracted with acetonitrile and interferences reniored n-ith charcoal. A modified bromophenol blue-silver nitrate chromogenic agent !vas used for final identification. Improved resolution was achieved by using two - dimensional chromatography.
Getz (29) also developed a paper chromatographic procedure to separate 18 products formed from three organophosphorus ester pesticides on kale. nlacRae and McKinley (59) have outlined two paper chromatographic procedures for separating some organophosphate insecticides. Ident'ification was by a technique baaed on the binding of ferric ions by the phosphate ekter grouping and reaction of the free ferric ion with salicylsulfonic acid. The phosphat'e esters appeared as white spots on a mauve background. Thiophosphates were converted to their oxygen analogs, on filter papcr, by exposure to bromine vapors. After bromination on acetylated paper, the thiophosphates appeared a.j intense yellow spots on a mauve background, whereas the phosphate esters appeared as white spot-the g m chromatographic. Tlie polarographic method was most rapid, the colorimetric method the mo?t time-consuming. The microcoulometric gas chromatograph allpeared to be a good compromise to give acceptable accuracy in a reasonable timc. Leinbach and I3rekke (60) have modified the Gibbs method for determining o-phenylphenol in fruits. A more efficient distillation apparatus is described, and by distilling from solutions buffered with calcium carbonate, suitable recoveries were obtained a t levels of 1 p.p.m. or less. A colorimetric method for determining dodine residues in plant materials has been developed by Steller et al. (82). After suitable est,raction and cleanup, the compound is complexed with bromocresol purple in a buffered, aqueous alcohol solution. The complex is then estracted into chloroform, then into aqueous alkali, and the absorbance measured a t 590 nip. The method will determine 0.2 t,o 2.6 p.p.m. in a 50-gram sample. RIcKinley and Nagarvey (66) have reported a method for determining Phygon (2,3 - dichloro - 1,4 - naphthoquinone) residues in cherries. Phygon react.ed with triethylamine to produce a colored product. Tlie absorbance was measured a t 672 mp and as little as 10 p g . corild be detected.
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ANALYTICAL CHEMISTRY
HERBICIDES
Herrett and Linvk ($8) have described a simple colorimetric method for determining 3-amino-1,2,4-triazole in biological systems. After extraction and isolation, the compound is diazotized, then coupled Rith 8-amino-lnaphthol-3,6-disulfonic acid in the cold. The sensitivity range, as used, was 0.1 t o 3.3 p.p.in. A satisfactory method for determining maleic hydrazide residues in tobacco was reported by Hoffman (39). Previous methods had been unsatisfactory for tobacco because of interfering colors. This interference was eliminated by collecting the distilled hydrazine in sulfuric acid Rolution and boiling it down before developing the color. Lane (47) has tlr~ve1ol)ed a colorimetric method for determining FalonP (tris - 2,4 - dichlorophenoq-et.hy1 phoaphite) residues in food crops. Tile Falone is hydrolyzed to produce 2.4dichlorol)lienouyetliaiinl, that is t,hen steam-distilled. Carbon tetrachloride is used to extract the conipound from the distillate and concentrated sulfuric acid eytrwts it from the carbon t,etrachloritlc. The method is based on the reduction of the intense piirple color of nitrated diinethylnaphthidine by the 2,4-tiichlorophenoxy conii)ound present. The procedure is app1icwl)lc to amounts from 1 to 10 pg. paper chromatogrwi)liic method for determining Atrazine (2-chloro-4-eth,vlamino-6-isopropylamino-s-triazine) and Simazine [ 2 - chloro - 4,6 - bis(ethy1amino) - s - triazine] in corn has been devcloped by Major (61). Chloroforin extraction from ammoniacal solutiuir was followed by separation and partixl cleanulj on an oxalic acid and'oi, perchloric acid-Celitc column, tlim :I final elennui, on a Florisil coliiiiin. Paper chromatography was tlieii used for identification. Marquardt and Luce (63) have developed a method for determining phenosy acid herbicides in agricultu1,al products. Xoting that previous methods were not, nlnays satisfactory, they cleaved the phenosy acids with pyridine hydrochloride, vhich released the phenol derivatives. These could be det,ermined colorimetricallj- a t concentrations as low as 0.05 p.p.m. in terms of original compound in the product analyzed. Cleanup n-as also simplified by this procedure. Tlie method for dcteriiiining residues of 3-niiiino-1,2,4-triazole, developed by the U.S. Food and Drug Adniinistration, was modified by Storherr and Burke (83). The modifications were in the cleanup procedures, which produced control readings for various crops from 0.003 to 0.02 p.p.m. The
method is sensitive t,o 1 pg. in a 40gram sample. Riden and Hopkins ('76) have developed a method for determining barbane residues in crops. Residues were hydrolyzed to produce 3-chloroaniline, which was steam-distilled and determined colorimetrically by diazotizing and coupling with S-(1-naphthyl)et hj-lenediamine dihj-drochloride. The method n-ill detect about' 0.02 p.p.m. in a 100-gram sample. d method for determining residues of diuron, fenuron, monuron, and neburon !vas developed by Dalton and Pease ( I S ) . The three azo dyes from aniline, p-cliloroaniline, and 3,4-dichloroaniline were separated and determined, permitting the identification of fenuron, monuron, and diuron alone or in mixtures. Seburon and diuroii both give 3,4-dicliloroaiiiline upon hydrolysis, and cannot be distinguished from one another in iilixtures. Aftcr hydrolysis, the resukiiig anilines were diazotized a.nd coul)lccl \vit,h .V-(1naphthyl) - eth>-lenediaii,iiic, tlih!drochloride and absorbaiim ~ c r nicnsrircd e a t 560 nip. Pease (73) lias Iiublished a ~)rocedurefor det,eriiTining monuron :ind diuron individually. in mixtures of the two. The azo d ! - ~were formed, then separated by a iiquid clironiatographic technique. An apparatus for continuous extraction of the stemivolatile substances is described. -4 paper chromatographic screening pro(w1urc for monuron, diuron, aiid neburon residues was reported by Major ( 6 0 ) . The herbicides were extracted in ammoiiiacvl solution with chloroform, transferred to petroleum ether, and then extracted withivater to remove monuron and diuron. The neburon remained in the petroleum ether. After part'itioning into appropriate solvents, a Florisil column was used for further cleanup and :Lliquotr ivcre spotted on paper. Mitchell (67') has described a paper chromatographic procedure that will separate 2,4-D and 2,4,5-T. Solvent systems, reagents, and R , values are givcn. MISCELLANEOUS
Major and Barry (68) have described a screening method for chlorinated hydrocarbons and malathion residues 011 fruits and vegetables, ivhich employs paper chromatography. Essentially a screening method for high levels ( 5 p.p.m. or more), a saving in time and solvents is achieved by cleaning u p the sample extracts on paper, with acetonitrile. Segal (79) has developed a colorimetric method for determining thiocyanate in corn, in connection with the use of Bmitrol-T (an equimolar mixture of 3-amino-1,2,4-triazole and ammonium thiocyanate). After aqueous
extraction and conversion to cyanogen chloride, microdiffusion separates interferences with simultaneous formation of color with a pyridine-pyrazolone system. As little as 0.2 pg. of thiocyanate may be determined in corn. A procedure for determining residues of Lethane 384 [ l-butoxy-2-(2-thiocyanoethoxy)ethane] in milk and animal tissues has been reported by Gordon, Haines, and Wolfe (33). Methods for e\;traction and cleanup of milk and animal tissues t o eliminate interferences were developed. The actual determination employed the known colorimetric method, but suitable conditions for hydrolysis of Lethane to inorganic cyanide had t o be determined. Hydrolysis at room temperature with sodium methylate and dimethylformamide proved satisfactory. Detection at a level of 1 pg. was possible. Bruce, Robbins, and Tuft (7') have developed a method for determining phosphine residues in wheat a t levels of 0.005 p.p.m. or less. The gas was absorbed in bromine water, the bromine boiled off, and a phosphate determination made on the solution. The method proved satisfactory over a range of 0.8 to 12 pg. of phosphorus. Procedures for determining Guthion and Sevin (1-naphthyl Ar-methylcarbamate) in tobacco and cigarette smoke have been developed by Bowery and Guthrie ( 5 ) . The existing colorimetric methods were used, but modifications in extraction and cleanup procedures were necessary to reduce the interferences in tobacco evtracts to a reasonable level. Berck (4) has proposed a method for determining methyl bromide, ethylene dibromide, and carbon tetrachloride in admixture. in air. The procedure is based on the kinetic differences in rate of hydrolysis with monoethanolamine. At 3" C., only methyl bromide reacts. At 100" C. ethylene dibromide and carbon tetrachloride may be measured by aigentoinetric titration. In mixtures, ethylene dibromide was determined by selective oxidation of the bromine in the hydrolyzate by a modified Kolthoff-Yutzy method and the iodine end point determined amperometrically . Alethy1 bromine was determined coulometrically by Dumas and Latimer (19). The vapor mas absorbed and converted to inorganic bromide in an alkaline alcohol solution. The minimum sensitivity was reduced to 3 pg. per sample by modifying the instrument. Miskus et al. (65) have presented a colorimetric procedure and a paper chromatographic system for determining aromatic carbamates and some common phenols. Alkaline hydrolysis of the carbamate insecticides produces aromatic moieties t h a t react with pnitrobenzenediazonium fluoroborate to
produce colored compounds. Phenols react directly with the reagent in acid solution. A paper chromatographic system for separating the carbamates is given. A method for determining Karathane [a mixture of dinitro(1-methyl-1ieptyl)phenyl crotonate (78%) and dinitro(1methylhepty1)phenol and related compounds (22%)] residues has been developed by Skerrett and Baker (80). The residues were hydrolyzed n-ith alcoholic tetraethylammonium hydroxide solution and the absorbance was measured at 425 mp. KO cleanup was required for strawberries but was required for apple leaves. Smith, Thiegs, and Swank (SI)have described a method for determining residues of Zoalene (3,5-dinitro-o-toluamide) in chicken tissues. The Zoalene is extracted with acetone and benzene, then chromatographed on an alumina column. A colored compound is then formed by reacting with 1,3-dianiinopropane in the presence of dimethylformamide. The limit of detection is about 0.1 p.p.m. in chicken tissue. Thiegs and Smith (85) have developed a method for distinguishing betm-een Zoalene and 3,5-dinitrobenzamide. This method involves forming the colored complex and measuring the absorbance at 540, 560, and 600 mp. The ratio of the absorbance a t 600 mp to that a t 540 or 560 mfi serves to identify the compound. This method can also be used to determine 3-amino-5-nitro-otoluamide after first converting to the dinitro compound with peracetic acid. A colorimetric method for determinin ing 3-amino-5-nitro-o-toluamide chicken tissues was proposed by Thiegs, Smith, and Bevirt (86). After extraction and cleanup, the compound is diazotized and coupled with A--(lnaphthy1)ethylenediamine dihydrocliloride to form a colored complex. Boyd and Barber (6) have del eloped a method for determining residues of tributyl phosphorotrithioate in cottonseed. The compound is liydrolyzed rvith sodium borohydride to liberate butyl mercaptan. The mercaptan is distilled rvith mercuric acetate solution and determined colorimetrically. The hydrolytic technique is new and should be applicable to other sulfiir-containing phosphorus esters. CLEANUP PROCEDURES
Almost all of the analytical methods for pesticide residues include cleanup procedures. The problem of cleanup is of sufficient importance, however, to justify some additional attention. Moddes (70) has investigated the various grades and activation of Florisil and established conditions for achieving maximum activity. Activation of 110" or 260" grade in a muffle furnace a t
650" C. for 1 to 3 hours gave best results. -4 procedure for standardizing columns n ith a dye mixture was del-ised. Johnson (42) developed a procedure for separating dieldrin and endrin from other chlorinated hydrocarbon pesticides. Extracts were placed on a Florisil column, other pesticides eluted with 6% ether in petroleum ether, and endrin and dieldrin then eluted with 15y0 ether in petroleum ether. An acetonitrile partitioning completed the cleanup necessary for quantitative determination in a microcoulometric gas chromatograph. Bates, Rowlands, and Harris (3) have devised a cleanup procedure for malathion residues in barley and rice bran. Chromatography on acid-washed alumina nas used for barley and on fuller's earth for rice. Adsorbent grades and elution systems are given in detail. A procedure for the cleanup of butterfat prior to dieldrin analysis was developed by NcKinley and Savary (56). Partitioning between acetonitrile and n-heuane vias followed by elution from a Darco G60-Solka Floc column with acetone. Samples thus treated may be analyzed by paper chromatography. McKinley, Savary, and Rebster (57) have devised a cleanup procedure for fat samples prior to analysis for D D T , D D E , and TDE. Fats were precipitated by freezing at 5" and -70" C. -4Florisil column was used to complete the cleanup. Moats (69) has developed a cleanup procedure for fats t h a t utilizes a silicic acid or a silicic acid--sulfuric acid column A 20-gram coluinn will handle a 2-gram sample of fat when silicic acid alone is used. The capacity of the combination column is about 1 gram of fat to 15 grams of the adsorbent. DDT, DDE, chlordane, heptachlor, lindane. and to\;alihene were run on the acid rolm?n. ;Ildrin ran he niii on the straight silicic acid column onlj.. A partitioning procedurp used by Eidelman (20) utilized dimethvlsulfoxide (DSMO) to separate chlorinated hydrocarbon pesticides from hittterfat. Residues a' lon as 0.02 t o 0 05 11 I? m. could be separated. Storherr and Onlcv (Si) h a ~ cdeveloped a dry-packed rpllulo~ccolumn for cleanup of n t r a c t s for h n i i n o 1,2,4triazole analvsis. The column can be re-\iced niany time?. Xo acetylation or crosq linking of the cellulose is required. By the selective use of polar and nonpolar solvent systems, the compound and it. metabolites may be separated. LITERATURE CITED
(1) Bache, C. A , Lisk. D. ,J. J . Agr. Food Chem. 8,459 (1960). (2) Baumler, J , Rippstein, P., Helv. Chim. Acta 44, 1162 (1961). (3).Bates, A. N., Rowlands, D. G., Harris, A. H., Analyst 87, 643 (1962). VOL 35, NO. 5, APRIL 1963
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(4) Berck, Ben, Canada Dept. Agr., Res. Branch, Pub. 1101 (1961). (5) Bowery, T. G., Guthrie, F. E., J . Agr. Food Chem. 9, 193 (1961). (6) Boyd, G. R., Barber, M. A., Ibid., 10, 196 (1962).
(7) Bruce, R. B., Robbins, A. J., Tuft, T. O., Ibid., 10, 18 (1962). (8) Burchfield, H. P., Storrs, E. E., [‘Biochemical Applications of Gaa Chromatography,” Academic Press, New York, 1962. (9) Burke, J., Johnson, L., J . Assoc. O&. Agr. Chemists 45, 348 (1962). (10) Calderbank, A., Turner, J. B., Analyst 87, 273 (1962). 1) C a d , C. C., “Residue Reviewe,” Vol. 1, p. 37, Academic Press, New York. 1962. 2) Cktro, C. E., Schmitt, R. A., J . Agr. Food Cham. 10, 236 (1962). 3) Caverly, D., Hall, P. S., Analyst 86, 478 (1961). (14) Chilwell. E. D.. Hartlev. G. S.. Ibid., . 86. 148 11961). ‘ (15) ’Cox, ’W.S., J . Assoc. Ofic. ilgr. Chemists 44, 188 (1961). (16) Ibid., 45, 406 (1962). (17) Curry, A. N., Kress, L. M., Paylor, R. A. L., J . Aqr. Food Chem. 9, 469 (1961). (18) Dalton, R. L., Pease, H. L., J . .4ssoc. Ofic.d g r . Chemints 45, 377 (1962). (19) Dumas, T., Latimer, R. A,, J . Agr. Food C‘hem. 10, 276 (1962). ( 2 0 ) Eidelman. )I., J . Assoc. Oflc. A g r . Chemists 45, 672 (1962). (21) Kidusoil, H, P.. Ibid., 44, 183 (1961). (22) I;rickson, L. C., Hield, H. Z., J . Agr. Food C h m . 10, 204 (1962). ( 2 3 ) Faliej.. ,J. E., Pchechter, M. S., Ibid., 9, 1!)2 ( 1 U i i l ) . 124) (;age. J. C.. ddvan. Pest Control Res. 4; i53 (1901). (25) Gainn, R. J., J . Assoc. Oflc. Agr. C h ~ , , / z > 45. l s 401 1962). (26) George, 1J. .\.,~Fahey, J. E., TValker, K. C., .I. S g r . Food Chein. 9, 261 (1961 1. (27) Gershman, L. I., J . Assoc. O f i c . Agr. Chemists 44, 212 (1961). (281 Gets. J. E.. Ibzd.. 45. 393 11962). (29j Ibid.; p. 397. 130) Giana. P. A., J . Aar. Food Chem., 9, 42 (ig6ij. (31) Goodwin, E. S., Goulden, R., Reynolds, J. G., Analyst 86, 697 (1961). (32) Gordon, C. F., Haines, L. D., Rosenthal, I., J . Agr. Food Chem. 10, 380 (1962). “
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(33) Gordon, C. F., Haines, L. D., Wolfe, A. L., Ibid., 9, 478 (1961). (34) Guinn. V. P.. Potter. J. C.. Ibid.. 10. . 232 11962,. ’ (35) Gunther, F. A,, Blinn, R. C., Carman, G. E., Ibid., 10, 224 (1962). (36) Gunther, F. A., Blinn, R. C., Ott, D. E., J . Assoc. 0.ffic. - AUT.Chemists 45, 359 (i962). (37) Gutenmann, W.H., St. John, L. E., Barrv. D. L.. Jones. E. D.. Lisk. D. J.. J . Agr. Food Chem. 9, 50 (196l).’ (38) Herrett, R. A., Linck, A. J., Ibid., 9, 466 (1961). (39) Hoffman, I., J . Assoc. Oflc. Agr. Chemists 44, 723 (1961). (40) Hughes, J. T., Analyst 86,756 (1961). (41) Hughes, R. E., Jr., Freed, V. H., J . Agr. Food Chem. 9, 381 (1961). (42) Johnson, L., J . Assoc. Oflc. Agr. Chemists 45, 363 (1962). (43) Kilgore, W. W.,Cheng, K. W., Ogawa, J. M., J . Agr. Food Chem. 10, 399 11962). (44)Klein, A. K., Gajan, R. J., J . Assoc. Oflc. A g r . Chemists 44, 712 (1961). (45) Koblitsky, L., Adams, H. R., Schechter, 31. S., J . Agr. Food Chem. 10, 2 (1962). (46) Kotakemori, hi., Hauda, H., A n n . Rev. Takamine Lab. 13. 260 (1961). (47) ‘Lane, J. R., J . .4gi. Food Chem. 9, 377 (1961). (48) Lausen, H. H., .Tuture 194, 1174 (1962). (49) Laws, E. Q.,Weblev, D. J., Analyst 8 6 , 249 (1961). (50) Leinbach, L. R., Brekke, J . E., J. Agr. Food Chenz. 9, 205 (1961). (51) Lisk, D. J., “Residue Reviews,” Vol. 1 , p. 152, hcademic Press. S e x York, 1062 ( 5 2 ) Lovelock, J. E.,
-4\41 CHEV 33, 162 (1961). (53) MacDonald, A. RI G., Analyst 8 6 , 3 (1961) (54) MacDouPall. D.. “Residue Reviews,” Vol. 1, p. -24, .i&dcmic Press, XPW York, 1062. ( 5 5 ) McKinle); W. P., Magarvey, S. A . , J . Assoc. Oflc. $gr. Chemists 43, 825 (1960). (56) McKinley, W. P., Savary, G., J . Aar. Food Chem. 10, 229 (1962). (57)”McKinley, W. P., Savary, G., Webster, C., Ibid., 10, 226 (1962). (58’1 ., McKinlev. W. P.. Savarv. G., Webster, C., J.’:4ssoc. Ofic. .49;. Chemists 44, 193 (1961). \
(59) MacRae, H. F., blcKinley, R. P., Ibid., 44, 207 (1961). (60) Major, A., Jr., Ibid., 45, 387 (1962). (61) Ibid., p. 679. (62) Major, A., Jr., Barry, H. C., Ibid., 44, 202 (1961). (63) Marquardt, R. P., Luce, E. N., J . Agr. Food Chem. 9, 266 (1961). (64) Mills, P. A,, J . Assoc. Ogic. A g r . Chemists 44, 171 (1961). (65) Miskus, R., Eldefrawi, M.E., Menzel, D. B., Svoboda, W. A,, J . Agr. Food Chem. 9, 190 (1961). (66) Mitchell, L. C., J . Assoc. Ogic. Agr. Chemists 43, 810 (1960). (67) Ibid., 44, 721 (1961). (68) Ibid., 45, 683 (1962). (69) Moats, W.A., Ibid., 4 5 , 355 (1962). (70) Moddes, R., Ibid., 44, 169 (1961). (71) Murphy, R. T., Barthel, W. F., J . A g r . Food Chem. 8, 442 (1960). (72) Ott, D. E., Gunther, F. A., Analyst 87, 70 (1962). (73) Pease, H. L., J . d g r . Food Cheni. 10, 278 ilR62I --,(74) Phillips, D. D., Pollard, G. E., Soloway, S. B., Ibid., 10,217 (1962). (75) Riden, J. R., Hopkins, T. R., Ihid., 9, 47 (1961). (76) Rusk, H. W.,Fahey, J . E., Ibid., 9, 263 (1961). (77) St. John, L. E., Jr., Lisk, D. J., Ihid., 9, 468 (1961). (78) Schechter, M. S., Westlake, W. E., Ax.4~.C m a r . 34, 25-4 (January 1962). 179) Seaal. H. S.. J . d q r . Food Chem. 10, I O 1lg62). (80) Skerrett, E. J., Baker, E. .I.,Analyst 87. 228 --- (1962). (81) Smith, G. N., Thiegs, B. J., Swank, pvl. G.. J . A g r . Food Chem. 9, 197 (1961 ). (82) Steller, W.A., Klotsas, K., Kuchar, E J.. Sorris. 31.I-.,Ibzd.. 8. 460 11960). ( 8 3 ) Ptorherr, ’R. W.>Bur’ke; J . , 2. Assoc. O ~ CAyr. . Chemists 44, 196 (1961). (84) Ptorherr, R . W.$Onley, J., Ibid., 45, 382 (1962). (85) Thiegs. B. J., Smith, G. S . , J . -.igr. Food Cheni. 10, 26 (1962). (86) Thiegs, B. J., Smith, G. S . , Bevirt, J . L., Ibid., 9, 201 (1961). (87) Wells, C. E., Bureau By-Lines (E. P. Dept. of Health, Education and Welfare, Food & Drug Admin.) 4, 67 (1962). (88) Westlake, W. E., ANAL. CHEJI. 33, 88R (1961). (89) Yip, G., J . Assoc. O f i c . d g r . Chemists 45,367 (1962). (90) Zweig, G., Archer, T. E., Raz, D., J . d g r . Food Chem. 10, 199 (1962). \ - -
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