LIEP.ARY p; , :>:t BOARD AvTil 1942 S-553 A PJSVISW OF METHODS lOR TH3 CH3lv:i GAL ANALYSIS OF ROTSWOIIS-BSARING PJ^ANTS ijy Howard A. Jones, Division of Insecticide Investigations CONTSilTS Page Introduction -------------------------- 3 Snjnpling ---------------------------- 2 Moisture ---------------------------- 5 Total extract ------------------------- 7 P-otenone ---------------------------- 14 Deg^jc^lin and rctenone plus deguelin -------------- 48 Toxicarol and other alkali-solucle s^^tstances --------- 59 ^'iscellan'^ous color in a trie determinations ----------- 61 Other determinations ----------------------- 56 Literature cited ---- -------_---___--_-- 71 - 2 « 4 i?ji^5duction An attempt has been made to collect all references to riethocs used for the cherdcal analysis of rotenone-bearing pl?iits, particularly of derris aiid cu"b3 roots. The co.iipilation nas "been corfined to articles descri-oing rnethcds or criticallv comparinfl: results obtained ty different methods. Iteferences {ri-?rinf: only results of ar^alysic or reviews ccntslning no original contributions or critical observations hav? net been included. Likewise methods of utilizing the results of chenical analysis, directly or t^irough various calculations, to obtain a nieasurs of insecticidal effec- tiveness are- not treated here. This problem is itself sufficiently dis- tinct and important to require separate treatment. The methods discussed here have been f::rcuped primarily accordinr to the subntances which they d^-Ptermine . This classification, although difficult because of uncertainty as to .iupt what substances are determined by many of the methods, was believed to be the most useful. At the end of the sections on methods for determining; the more important constituents, a brief statenient has been mads of the present status of methods for the purpose. In the last tvo sections are grouped certain methods that give more or less empirical values, which it is difficult or imoossible to interpret in terns of actual substances present in tne sajcple , SAtrpLING In this section only those few articles are reviewed which give specific details on the s.amplin^ of derriv; or cube root for analysis. Georgi and Teik (74) in 1933 surgestsd a procedure to be followed in selecting a sample for analysis from a s'iipmriint of derris root. In 1936 these authors (35) elaborated the method somewhat, so that, for the preparation of fresh roots, the proced'ore was as follows: "Fresh roots are sun-dried until they can be broken without ey:udation of plant -.juices. Th^ serai-dried roots are then c":t into pieces ^'arying from l/4 to 1/2 inch long and the chopp<*d naterial is further sun-dried until of constant wei^^ht. The j-'terial is then quartered for analysis until s.r)proximately 1/4 lb. remains. One h\indred grarr.s are v/oighed, then ground in a drug mill. The root is first passed througn a disint-^grator having spaces l/32 inch v;ide, the spaces bein^ appror.ir.ately 1/4 inch apart. Tho naterial is .gro^iond a second tim-:; , the dis- integrator being r-'^placed by a 1 mm. sieve. The srr;ail anount of woouy matter reniaininr is ground separately in a small mill and incorporated with the main ssmnle . To obt^-in a representative sample of the ground root for analysis, the varicii.s fractions are shaken together in a .iar provided v;ith a closoly-fitting lid, and the required amount of material io weighed before segregation of the fine particles h^s taken place." In sampling small shipments it was proposed thatl pound of roots "be withdrawn at random from a tals of 200 po^inds. In the cas« of a lar^^e shipment it was suggested taat 10 perceiit of the "bales "be sani-nled and 5 percent "by v/ei:;ht of each of these "bales "be removed. The roots v;ere to be chopped into short lengths and quartered until 1 poi.ind remained. Cahn and Bo am (13) in 1955, in an article on the determination of rotenona, made the following: statement in regard to sampling: "Derris root should pass entirely through a 50-mesa sieve "before it is sampled. Eoot, as ordinarily f;round, frequently con- tains a proportion of coarse, spicular material, through which the pov/der (often extremely fine) tends to filter, thus ceusinf:; an undue amount of coarse particles to "be present in' tne top layers. If the a-'^ount of sioicular material is considerable, the whole baton should "be sieved- through a 50-mesh sieve, ard the samples taken from proportionate aT.cunts of tne fine and coarse n'aT^erial . " Gahji (JL2) again in 1936 stressed the necessity of proper sampling. He found that, of 10 "bales analysed cut of a consignment of 40 "bales from the same estate, the total extract ranged from 15 to P5 percent. The only satisf actor;.'- method of sa.mplin.e derris root was said to "be to grind it first, mix i^ thorough'.ly , arc then anal'-ee the mixed fii^e powder. Gecrgi (Sl) in 1937 described a method used in sampling kiln-dried root during the process of "baling. Vrnen half the quantity required for a "bale had "been loaded into the press, a"bout c. pounds of the root v/as taken at random from the heap and set a,side as a saraple from that "bule. When 20 camples, say 40 rounds, ha,d ."ccimulated, th^^ bulk sample of root was mixed, spread cut in a thin layer .on the floor, an'-"' qua^rtered until a"bout 1 pound remained. This amoimt was cut into 1-inch lengths and quartered until about 3 ounces remained. This quantity was further cut into 1/4-inch lengths and ground in the la"bcratory mill for ar^alysis. In one ca.se 25 samples covering 497 "bales v/ere analysed. In 1937 Levallois (62) stated that the sampling of rotenone- "bea^ring roots was very delicate, as the rotenone ■ content of a sample depended on the a'Dunn^nce of long fibers. Progressive 5:rinding of roots gave fractions with rotenone contents as follov/s: Fria"tle parts and short fi'ijers 6, mediiim parts 4 to 4.5, and long ii"bers 3 to 4 percent. Tnis author did not take samples of derris or cube at random, but classed root in three or four categoriea according to size and sampled accordingly. From each lot he took an aliquot part of l/lO to 1/20 and chopped, up these parts. From each pa.rt he took a new fraction. These fractions miide up the samples for analysis. Chevalier and Chevalier (20) recommended that, since large and small roots differ in rotenone and total-extract conte'nt, samples taken for analysis should have a'bout the same proportion of large and small roots as the whole lot. - 4 - Kriikoff and Smith (80) in 1937 reDorted that, if a lot of crude Loncho carpus roots is a mixture of different species, no sample will accurately represent the entire lot. In sampling large, untaled lets of a single species, fairly accurate results were obtained by making a care- ful ocular estimate of the weight of small, medium, and large roots in a given lot and taking a corresponding sample. For smaller lots, such as roots from a single plant, the different sizes were sorted out, weighed, and the sample was taken accordingly. This method would be impractical for sampling baled shipments as they arrive in this country. In the pre- paration of samples of fresh roots for analysis more uniform results were obtained by dr-ying them to about 12 percent moisture before reducing them to slices or shavings. Guillaume and Herve (53) in 1939 emphasized the importance of obtain- ing a representative sample in the analysis of plants containing rotenone. They suggested that a sample of at least 1 kg. of root be taken for p,Tinding. The ground material should be mixed and sifted and then mixed again at the moment of sampling. The Puerto Rico Experiment Station ( 123 ) recently reported that the already difficult problem of sampling baled derris root was further com- plicated by the variation in rotenone and total-extract content of roots of the same diameter found in worK at this station. It was suggested that a representative sample of the baled product could be obtained by using a small tube with a rotary cutter at the sampling end. Such a tool would make it possible to take a fairly large number of small samples and thus remove from the bale the minimum amount of root material required for a reliable analysis. Information on sajnpling was recently furnished by four large import- ers of derris and cube root: McCormick and Company, S. B. Penick and Company, John Powell and Company, Inc., and Derris, Incorporated (private communications). These firms agreed on the matter of sajr.pling about 10 percent of the shipment, although S. B. Penick and Company stated that frequently half, or even all, the bales in a shipment were sampled. John Powell and Company stated that where possible they preferred to grind at least one-tenth of the shipment. Two sources described the methods employed in removing samples of whole root. Derris, Incorporated, stated that 10 percent of the bales were opened and small bundles of root picked out at random from various sections of each bale. This material was ground, mixed, and a l-pound sample drawn for testinrf^;;. McCormick and Company removed from each of the bales chosen three small bundles of root, and from each bundle chopped sections about 4 inches long from the fine ends, the large ends, and the middle. These sections were used to make a composite sample for analysis. Discussion The sampling of derris and cube roots for analysis is verj-- important. The proper sampling of whole root is extremely difficult, and it is doubted if any method is entirely satisfactory. In addition to differences in rotenone and total-extract content of different rrots in a shipment due to differences in plant source, it has also been shown that fine and coarse - 5 - roots of both derris and cube vary in t-^.eir content of rotenone and total extract (32, 33, 63, 64). Hence, in i:.pimpliT.g. a whole-root shipment care aust bs taicen not only to draw froc different parts of the bale or case, but also to obtain a sample havin?; approximately tne same proportion of fine and coarse roots as does the whole s'lipmsnt. This proce^vre v/as recorajnended by Chevalier and Chevalier (20) arid by Kriikoff and £:r;ii,h (60) . The method cited above as used by one Ajnerican importer is an attempt to accomplish this. The proper saxplihg of powdered root is no less important, although less difficult. Cshn and Beam (2.^).and other workers have pointed out that marked .se^sgat ion of fine and coarse particles occurs on standing. In addition to Levallois (82) and others, Koolhaas and Meijer-*- have shown large differences in the rotenone and total-extract content of fine and coarse powder from a single sample. They found in a sample prepared by them that the material passing an 80-mesh sieve analyzed 9.8 percent of rotenone and 19.6 percent of total ether extract, while the coarse material, which did not pass the sieve, had 1.3 percent of rotenone and 4.0 percent of ether extract. This variation indicates the importance of ccrrect sampling of powdered root, particular l,y material that has been subjected to shipment.-, IvrOISTUES In this section are discussed only procedures for the determination of moisture. Methods of dn^ing the sample before extraction, v/hich are not actual moistiire determinations, ar<3 discussed in connection with the various rotenone and total-extract methods. Tattersfield and Pvoach (122) in 1923, in determining moisture in samples of Derris ellipti ca, dried the materials to constant weight at the temperature of boiling dichloroethylene (55°-58° C.) in a partial vacuum over phosphorus pentoxide. Elevated temperatvj-e was avoided, as tne powdered root was said to decompose on prolonged heating. A similar method was used by Georgi and Cutler (32) in 1929 in analyzing derris roots. Spfon ( lis ) in 1931 determined moisture in derris by heating 5 grams of the gr«>und root at 100^-102° C. G-e©rgi and Teik (34 ) stated in 1933 that moisture determinations might be made either by drying the finely ground material to constant weight in a steam oven at 100° C. or by distilling with xylene. In the former method a 5-gm. sample v;as used, while in the distillation method 40 to 50 gm. was employed. The distillation method was preferred, as it was said to eliminate oxidation during drying. Report of the analysis of a sample of derris root by various laboratories. 19 pp., typewritten. Jan. 19, 1937. - 6 - lattersfield and ^lartin (120), in their work on evaluation of roten- cne-containicg plants in 1935, reported determinations of moisture "by these ■two methods. The xylene-distillation method was used in.I.'alaya, while the same samples were analysed for moisture in 3ngl-:xnd.'oy drying at lOOP C. Higher results v/ere- ©"btained in ?'alaya, but the 'author? at tri'cuted the dif- ference to greater absorption of atmospheric- rr^oistva'a there.- = Again in 1976 C-eorgi and Teik {c§) stated thatimoisture was best determined by the xylene-distillation metrxd, but could elso be det5rm:ined by drying to constant weight at lOC^ 3. Koolhaas ajnd Meijer^ in 1937, in a report on.-the analysis of a sample .of derris root by various laboratories throughout the world, gave the fol- lowing results f^r moist'ore as oetermined by. the several laboratories: ........ Moisture Laboratory ■ Method •- • ■■ Pj^rcent A— United States Department of Airriculture Dried at 103-104° C.. .5.1 (Jones) ... . -fer •<; .hours .. -^ , ,. B — Department of Agricultiare , Straits Set- Xylene distillation • .11.3 tlementf, and Federated I'alay States (3-eorgi) C — Commercial J'useum of the Colonial Institute, iUnsterdam (i^owaan). D — The Cooper Tec^'^xical Bureau, London (Cahn and Boa,m) E— Iiothsijneted Experimental Station (Tat- ter sfield and Martin; F~Seil,. Putt, and Eusby, New York (Seil) G — Caeser and Lorentz, Halle-Saale, Germany H — Diethelm, Ltd, , Singapore I — Laooratcry for Chemical Research, Bui ten- 2,ojgv Java (Koolhaas and Ivlei.jer) constant weight Xylene distillation 10.4 Dried at 100° C 9,4 Dried at 100° C . 9.3 Ivot knoivn - 9.9 Over calcium chloride 5.1 for 2 days Dried ---t 85° 0. 7.2 Dried at 105° C,' 9.6 2 See footnote 1 - 7 - They concluded that itio.isture determinntioa siould 'b'' made by dryin.^ a 3- 2711, s.ample at 105° C. to consta.nt weight. The xylene or heptane method gave higher values, but the higher teir.perature to which the material v/as exposed was thought to leacV to decomposition, and the method was not recc-niiiended. Jones and Gr&haa (71) in 1938, in the ana.lysis of a large number of derris, cube, and timbo roots, determined moisture by drying 2-f^. sa-p.-'les at 106° C. for 2 hours. Two days' additional drying of some of the samples caused no signifibant additional loss in weiirht. Results for moisture content of oerris root by heating in tue oven and by toluene distillation v;ere compared in a repo:^'t of collaborative work by the Imperial Institute and the xtothamsted Experimental Station (5S) . Values bytne oven method were only slightly lov/er than those by the dis- tillation method. Meijer and Xoolhaas (89). in 1940 determined the- moisture content of powdered derris root by drying a 2- .to 3-gm. samtile to constant weight at 105° C. Of some interest in connection with the determination of the moisture content of derris powder is the hygroscopicity of the material. Meijer (87) in 1938 studied this question by keeping samples at different relative humidities at approximately room teinpsrature and neasijuring the increase in weight. After standing at 75 percent relative humidity roots originally having 6 to 8 percent of moisture were fouxid to contain 11 to 12 percent of moisture, while at 95 percent relative humidity these roots had about 24 percent of moisture. He concluded that pov/dsred derris root was "freely hygroscopic. " Discussion It may be concluded from the references given that the most suitable method for determining moisture is the drying of a 2- to 5-gm. sample at about 105° C. to constant v/eight. This should require only a few hours in maxLy ca,ses, but overnight d.rying before the first weighing should be a good practice. TOTAL HJXTHACT 'The determination of the total-extract content of derris and cube roots, although a com.paratively simple procedure and one that has been made from the time of the earliest work on these materia,ls, is nevertheless sub.ject to consid.erable variation. Tattersfield and Ecach (l22) in 1923, in the course of an investi- gation of Derris elliptica, pointed out that the following factors are to ' be considered in the determination of total extract: (1) 'The extraction solvent must be selective. (2) The root must be ground to sn almost impalpable powder. (3) Extraction must take place at fairly low temperatures; otherwise sr)arin£rlv soluble cnmnounds are formed. - 8 - (4) The extract undergoes chemical change on drying. (5) The type of extraction flask may modify results. (6) The extract is freed from the last traces of solvent with great difficulty. They proposed the extraction of the root in a Soxhlet apparatus, using a flat-tottomed flask, with ether (dried over anhydrous calcium chloride and sociium) , and drying the extract as rapidly as "008311)16 to constant weight of 100° C. A similar method was used ty Georgi ard Curtler (32) in 1929. They also pointed out that slight decomposition took place on drying the extract, and the results were slightly low. In the 1929 report of the Colonial Institute of Amsterdsun (77) it was stated that when a large sar.ple of root had been ground once to a coarse condition, a portion of this sample ground ac^rain to a finer stats, and some of the latter ground a tnird time to a very fine powder, the amount of ether extract was found to "be higher as the root was more finely ground. Dodwell and Company (private communication) in 1930 outlined the method in use in the trade for determining total extract of derris root. Ten grajDs of powdered root was dried in a vacuum over sulfuric acid and extracted exhaustively in a Scxhlst apparatus with dry ether, free from alcohol. The powder was then reground in a mortar and extracted. for an additional 4 hours. The extract was dried for about l/2 hoxir in a water oven. Later Dodwell and Company, (private communication) quoted a revised method for total extract in which the root was dried at 100° G. to constant weight and then extracted with dry, alcohol-free ether until exhausted. No second treatment v;as used. The extract was dried to constant weight at 100° C. Total ether extract was determined by Spoon (115) in 1931 by extract- ing 5 gm. in a Soxhlet with absolute ether for 15 hours and drying the extractives at 100'-0-102° G. K^oolhaas's (76) ether extraction method for rotenone included the determination of total extract. The filtrate and washings from the rotenone separation were evaporated, seated at 80° C. in a vacuiom for 1/2 hour, dried in a desiccator, and weighed. Their weight was added to that of the crude rotenone. The method for rotenone proposed by Jones (62) in 1933 and involving extraction of the root with carbon tetrachloride included a procedure for total extract, by which the filtrate from the rotenone separation, upon evaporation, was dried 1 hour at 105° C. and its weight added to that of the separated rotenone. - 9 - Georgi and Teik (34) in 19.33 compared other solvents with ether for the determination of total extract of derris root. FetroleTom ether was unsatisfactory, since its solvent action was so low. Acetone gave at higher value than ether. Slightly higher figures were obtained with chloroform, and slightly lower results v/ith carbon tetrachloride than with ether. Preliminary drying of the root did not change the amount of ether extractives' obtained. In a comparison of chemical composition and toxicity to insects of rotencne-bearing plants Jones, Campbell, and Sullivan (§9) in 1935 deter- mined total carbon tetrachloride extractives of several samples of derris and cube by the method cf Jones (62). Acetone and benzene e::tractives were prepared by Soxhlet extraction of 10- to 20-fTn. S3.vjples for 7 to 8 ho^lrs. In general the amount of materia-l extrsctec' v/ith acetone was higher than that with carbon tetrachloride, while that v;ith benzene was slightly lov;er. Tatter sfield and I'artin (l^0_) in 1935 gave results of the determina- tion of total ether extract on several samples of .derris. In the extrac- tion of 5-gm. samples v/ith ordinary ether slightlj'- higher results were ob- tained in Malaya than in Snfland. The results that voce obtained with sodiura-dried ether in England were slightly lover . than those obtained by the use of ordinary ether. In proposing their trichloroethylene extraction method for rotenone, Cahn and Boam (13) in 1935 stated that total extract could be determined by evaporation of the trichloroethylene solution to dryness and heating to constant weight in an oven at 100° C. In 1936 Georgi and Teik (35) gave directions, for the determination of ether extract of derris root. Five grams of the finely pov.'dered root was extract-od with ether in a Soxhlet for 16 hours. No appreciable addi- tional extract was recovered when the marc was regrotmd with sand and. again extracted v/ith ether. The ethereal solution was filtered if necessary to remove tiaces cf suspended matter, the solvent distilled off, and the residue dried to constant v/eight in a steam oven. Constant weight was usually reached in 6 hours. Mei.jer (85_) in 1936 proposed a colorimetric method for the approxi- mate determination of the total extra,ct of derris root. Tliis method is described in the section on Miscellaneous Colorimetric Determinations. V-'orsley (132) in 1937 determined total ether extract by extraction of a 10-na. sample in a Soxhlet for 24 honors and subsequent drying of the extract to constant weight at lOO" C. C-uillaume and Proeschel (54) in 1937 determined total ether and ace- tone extract of derris and other plants by treatment of 5-gm. samples in a specially designed continuous oercolatcr. Only 150 cc. of solvent was required, and the extraction v/as said to be complete in 12 hours. Ex- tractions with chloroform and carbon tetrachloride were made in a contin- uous percolator of different design. All extracts were filtered, con- centrated to about 20 cc, placed in the refrigerator until the solvent . - 10 - evaporated, and then dried at 50° C. for 12 hours and weighed. Valiifis for acetone extracts were highest, those for ether and chloroform lower and about equal to each other, and those for carbon tetrachloride generally- lowest. Koolhaas and Mei.jer in 1937 reported on the analysis of a sajnple of derris root by several laboratories in various parts of the world. Some of the methods and results for. total extract were briefly as follows: Laboratory Total extract Method (moigture-fr2e basis) Percent : ' A — United States Department of Agricultiire (Jones) 25 ^. benzene, 2 periods totaling 24 hours 18.2 E — Department of Agriculture, Straits Settlements and Fed- erated Malay States (Cxeorgi) 5 gm. ether, 18 hoiirs 19.3 C — Commercial Museum of the Colonial Institute, Amsterdam (Hov/aan) 5 gm. ether, 48 hours 19.1 D — The Cooper Technical Bureau, London (Cahn and Boam) 10 gm. ether, 3 periods totaling 96 hours 18.0 3 — Jnothamsted Experimental Station (Tattersfield and Martin) 5 gn. Analar ether, 3 periods 18.9 totaling 30 hours 5 gm. anhydrous ether, 2 periods, totaling 16 hours F — Seil, Putt, and Husby, New York 5 gm. ether, until exhausted (Seil) 17.7 19.2 G — Caeser and Lorentz, Ealle-Saale, 5 gm. ether C-ermany 18.6 H — Diethelm, Ltd., Singapore I — Laboratory for Chemical Re- search, Buitenjorg, Java (Koolhaas and Meijer) 25 gm. ether 18.1 50 gm. ether, 2 periods 19.6 totaling 48 hours See footnote 1. - 11 - The authors also reported their ovm results on 81 Scunples of derris. Results using ether were in close agreement with those using benzene, "but the , time required for exhaustive extraction with ether was 2 days and 3 nights, whereas benzene required only 36 hours. In discussing the determination of total extract Mei.jer (86) in 1937 recommended slow percolation of derris powder with ether in a Soxhlet. This was accomplished by placing the powder in the extraction tube of the apparatus without using a thimble. He also stated that drying of the extract, after removal of most of the solvent, was better accomplished at 40° C. under reduced pressure thaji by the usual drying at 100° C Jones and Graham (7l) in 1938 determined total benzene extract by Soxhlet extraction of 5-gm. samples. ..,.....-...;■;■' The determination of total-extract content was studied by Jones and Sullivan (76 X in 1Q38, in an endeavor to select the solvent and method that would most, readily extract all the toxic substances, from derris and cube roots together with the least amount of nontoxic material. The total- extract content of several samples was datermined by several procedures with various solvents. Successive extracts of some of the marcs with acetone, methyl alcohol,. and water were tested against mosquito larvae. In 7-hour Soxhlet extractions of 5-gm. samples the percentages of material extracted by benzene, carbon tetrachloride, and ether were generally lower than by .chloroform, ethylene dichloride , trichloroetnylene , and ethyl acetate, which gave values of about equal magnitude. Acetone generally extracted more material and methyl alcohol considerably more than did the other solvents. When samples were extracted with ether or benzene for an extended length of time, the amount of material removed agreed with that extracted by chloroform.- Acetone extracts of marcs from extraction with chloroform were in general nontoxic. I.'ethyl alcohol and water extracts following this were completely nontoxic. Acetone extracts of marcs from benzene and ether .extractions were toxic. Thus, of the solvents tested, chloroform appeared to be -.the most satisfactory from the standpoint of selective extraction of the. toxic material. Results by the multiple- extraction procedure and by the aliquot procedure with chloroform at room temperature were in agreement with those by ^oxhlet extraction. Because of its convenience, particularly when rotenone wag to be determined by the same method, the aliquot procedure v/as suggested as the most suit- able for determination of total-extract content! Rowaan and Van Duuren ( 108 ) in 1938 suggested chloroform extraction at room temperature for the determination of total-extract content of derris and cube root. Mei.jer (87) in 1938 showed the effect of drying the sample at elevated temperatures on tne amount of ether extract obtainable. 'Pnus, a sample of powdered derris root which before being heated contained 23.6 percent of ether extract gave 19.1 percent after being heated at 60° C. for 2 hours and only 14.4 percent after being heated at 80° C. for 2 hours. He concluded that heating above 50° C. before analysis should be avoided. - 12 - Eraak (9) . in 1939 rspQi*ted on the analysis of a sam'Bl^' oif dsrris root anal^/zed by the Laboratory for Chemical Research in Java; by Seil, Putt, and Rusby in New York; and by Salamon and Seaber in Loiidon. The three laboratories obtained almost identical values for the' ether- • .' 's extract, content , .. , . In collaborative work carried out between the Imperial Institute' and Rothajasted 3zperimentai Station (^) the ether-extract contents of three samples of der^-is root were determined. Values obtained at the former laboratory, where a 16-hour extraction was made, were about- 1 per- cent higher than those obtained at the latter station, where ' two 6-hour ' '' periods were used. By the latter scheme little additional extract was obtained in the second &-hour period except in the case of a high-rotenone root, which gave 1.4 percent additional material. All values ranged from 25 to. 30 percent, of total extract. i'eijer and Koolhaas (89) in 1940 described th^ method used in •"-- tneir laboratory for determining rotenone by extraction of a 50-gm. sample . for '65 hours with ether. The determination of total-ether extract was made in conjunction v;ith this as follows: "The ether is distilled off (from the mbther liquor from rotienone separation) on a water bath and the last trapes are removed in, a vacutim in a water bath not exceeding 40° C. The contents of tie flask are blown up to a voluminous mass, and placed in a desiccator over lime for 2 days, after _ which time constant weight has been reached. "The difference in weight between the fl'ask with resin and the empty, flask gives the amount of resin. To this the amount of crude rotenone is added, giving the ether extract in 50 g. of the sample..." These authors stated that the total-extract content was about the same when benzene, chloroform, or. ether was use."; hence the extra determination of ether extract was unnecessary when chloxciorm or benzene had been used as extraction solvent. A scheme of automatic hot percolation was described by I^.artin (83) in 1940. The apparatus consisted of a glass liner fitted into a wider glass oube connected with a 250-ml. flask and a condenser. The powdered root was supported in the liner on a pad of cotton wool, and rapid extrac- tion at the boiling point of the solvent was said to be effected. The percentages of resin extracted oy this method with several solvents from 25-gm. portions of a sample of D erris elliptica were determined. tie addition of petroleum ether. Spoon ( 115 ) , in the determination of rotenone in 12 samples of derris root, utilized cr.^ stallization from ether by Hoark' s method. In 1931 Jones (61) suggested the use of carbon tetrachloride to replace ether in the analytical extraction of rotenone from derris and cube roots. '3ther often gave extracts from waich it was difficult to separate rotenone. Carbon tetrachloride e:< tracts gave a quicker and more selective separation of the rotenone. In several cases in which no rotenone could be separated from the ether extract, carbon tetracnloride extracts separated rotenone readily. The rotenone separated from solu- tions in carbon tetrflchloride as a. solvate containing 1 mole of the sol- vent to 1 mole of rotenone. Blackie (7) in 1932 described an apparatus with groundglass .joints and mercurj'- sealjs, which was especially designed for the determination of the rotenone content of a Fijian plant b:'' extraction, wita ether. In the same vear Elackie (&) published results obtained for the rotenone con- tent of Derris uliginosa -ising tais apparatus and extracting for 36 .lours with ether. Georgi and Teik (33) in 193? described a r.iodif ication of P-oark's ether extraction method. They dried a 100-g^. sample in a water- jacketed vacuum drying oven at approximately 75° C. and then extracted with ether. The extract was concentrated to 40 cc. and crystallized in the cold cabinet overnight. In the sa-ne. year x-ool.haas (78) discussed the methods for the esti- mation of rotenone then in use. Tne ether extraction methods in particular were said tc be subject to error from such sources as insufficient fine- ness of root, too large a sample so that on evaporation of the extract the other substances present exerted an appreciable solvent effect, difficulty in wasning the rotenone free of impurities, and difficulties encountered in the filtration of viscous ether solutions. He proposed an ether extrac- tion method for derris root designed to overcome these objections. - 16 - As a measure of the purity of the crude rotenone the melting point was deterr.ined and, by means of a curve plotted from the melting .points of mixtures of various percentages of residual extract and pure rotenone, the amount of pure rotenone in the crude crystalline material was obtained. This method with few modifications ".as been used in the Butch 3ast Indies since that time. The modified metiod now in use is (described later in this section in connection with the more recent article by Mei.jer and Koolhaas (89). In 1933 Jones (62) described a method of assaying derris and cube for rotenone usin? carbon tetrachloride for extraction and crystallization. The plant material was extracted in a Soxhlet with the solvent, and the extract concentrated and set aside to crystallize. The rotenone separated as its carbon tetrachloride solvate , containing equimolecular proportions of rotenone and solvent. The extract was cooled in ice, and then the crystals were filtered throug-^. a Gooch crucibla and washed with ice-cold carbon tetrachloride. The crvstalline material was dried to constant weight at room te-'iperature and weighed. For more rapid but less accurate results the extract was cr-"-8talli2ed in an ice bath for a few hours and the solvate, after separation, dried in an air draft. This early method has been superseded by the method of Jones and Graham (72), from which has been evolved the official method of the Association of Official Agricultxir- al Chemists (2), which is given in detail later in this section. If the sample contained over 5 percent of moisture, it was suggested that it be air-dried at not much over room temperature before extraction. Lower results for rotenone were obtained when saTiples of derris root were dried at 100° C. in a vacuum for 5 aours. Overnight (17 nours) extraction was recommended for most samples, as it was found that some samples giving 6 to 10 percent of rotenone in 8 to 10 hours gave results about 1 percent higher when extracted for 15 to 17 hours. As a means of checking the purity of the separated rotenone solvate, neating to drive off the carbon tetrachloride of crystalli7ation and weighing the resulting rotenone were suggested. A deterrination of the optical rotation of the separated material was stated to give an indication of purity, while the chlorine and methoxyl contents were also suggested as of possible value for this purpose. The separated crystalline material was examined qualitatively to make certain that it was rotenone. This was done by microscopic examin- ation and melting-point deternination of the material recrystallized from amyl acetate. Several samples that gave no rotenone by the earlier ether extraction and crystallization procedure gave as much as 2 percent by the carbon tetra- chloride method. The latter method was believed to give correct results with roots containing over 0.5 percent of rotenone but with roots contain- ing 0.3 per cent of rotenone or less it was inaccurate. For such ma.terial it was suggested that large samples be used, a scheme involvinr extraction with acetoiie and crystallization of the extract from carbon tetrachloride was tried. Although acetone ^:ave a more rapid extraction of tae rotenone, much of the acetone extract was insoluble in carbon tetrachloride. The additional manipulation necessar?' resulted in a less pure product. - 17 - Also in 1933 Geor^i and Telk (34) stuc'ied the extraction and crystal- lization of rcterone .arid sngfested a. method involving-- the wt^s of carlicn tetrac^-loride. They 3U,?sested n edifications to- the ether extraction meth- ods of both Roark (lOO) ar.d Pr.oolhaas (78.) but; considered that ev.->n v/ith the modifications thep.e nethods did not gi-"-e satisfactoiy results. One change introduced in'P-oark's metncd involved drying the root in a vacuum at 75° C. for 6 hours. They stated that the drying did not, chanf;e the amount of extractives ohtained, but believed that the presence of mcisture interfered with the crystallization of the rotenbne. The. method surcrssted by these authors for estimatinf? rotenone uning carbon tetrachloride is briefly as follows: Fifty grams of the finely-ground root is treated for 72 hours (three active 8-hcur periods) in a Soxhlet with, carbon tetrachloride. The extract is concentrated until the solution begins to tnicken, and is allov/ed to cool and reeded if necessary. .The flask is allcwad to stand in the cold cabinet for 24 hcJirs. The crude solvate is filtered on a tared Gooch crucible and washed with 10 tp 15 cc. of ice-cold carbon tetrachloride. After standing in the air for 24 hours it is v;eit.hed. The filtrate from, the first crystallization is concentrated, cooled, and seeded. It is alDowed to stand in the cold cabinet overnight ar.d any additional crystalline material treated as i.iath the first crop. The total crystalline r -material is. treated with toiling alcohol, the solution cooled, and the separated: rotenone drieii at 100'-^ C. and weighed. ^ correction is rpade for the solubility of rotenone in alconol. The purity of the recrystallized. product is criecked by melting- point determinat-io'n. ' ' ~ These investigators tried acetcn'? and chloroform for ertraction followed by crj'-stallization from carbon tetracriloride . .The separatior^ „oi carbon tetrachloride-insoluble resinous material in the acetone extr.^cts inter- fered with the separation of the rotenone, and the use of f.iis solvent was not recommended. Chloroform proved satisfactory and extracted the rotenone in less time than did carton tetrachloride, but anV- saving of. time over direct treatnent with carbon tetrachloride seemed d.oubtful. The rotenone values obtained by the chloroform and carbon tetrachlbride methods were in close agreement. The- values by tha modified othe.r methods' were in general lower and were irregular. C-eorgi (30) in 1933 stated that carton tetrachloride had been substituted for ether in the estimation of rotenone. Spoon and Eov/aan ( 116 ) in 1933 described the method used by them for determining rotenone in derris root. This v;as essentially the method of Eoark with some modifications, such as longer crystallization and deter- mination of the melting point of the separated rotenone. The latter step was designed, not as a means of calculating the purity of the material as in the method of i^oolhaas , but as a checK on its identity and approximate purity. A procedure proposed by Takei, L'iya.jima, and Ono ( 118 ) ir 1933 was a close approach to a truly chemical method for rotenone. Since the method - 18 - was designed to determine both rotenone and deguelin, it will "be discussed only briefly here and described in more detail in the section on Depruelin and fiotenone Plus Deguelin. The root sample was extracted with ether and after crystallization of a first crop of rotenone from the extract the evaporated mother liquor was oxidized so that there were formed dea;-dTo- rotenone, from the remaining rotenone, and dehydrorieCTielin. The mixture of insoluble dehydro compounds was separated and weighed. It was then subjected to catalytic hydrogenation under suitable conditions, when the dehydrorotenone was converted to the alkali-soluble isodihydxoder^dro- rotenone while the dehydroc'e^Tuelin remained -unchanged, thus in effect giving separate values for rotenone and deguelin. Some (?erris-root samples gave s-urprisingly large amounts of additional rotenone by this method. In one extreme case only 0.56 percent of lotenone was obtained by crystallization, while an additional 4.74 percent was obtained by the chemical treatment of the n^other liquor. On the other hand, in a sample giving 6.38 percent of rotenone b-" crystalli-^ation only 0.52 percent of additional rotenone was obtained. Values or. some other sajnples ranged between theee extremes. One sample giving no rotenone by ether crystalliz- ation gave 0.97 percent by the chemical metnod. Later work by Tatters- field and ilartin (120) , however, has shown that the ether crystallization used by Takei and coworkers was inco^iplete. In the same year Danckwortt and Budde (24), in an investigation of methods for the evaluation of derris root, proposed the separation of rotenone by extraction with chloroform followed by crystallization from ether. The powdered root was covered with 10 times its weight of chloro- form, and the mixture allowed to stand for 24 hovrs with frequent agitation, An aliquot of the ciloroform extract equivalent to six-tenths of the original sample was filtered, the chloroform removed, and the residue crystallized from ether. In 1934 DancKwortt, Budde, and Baum^rarten (25) reviewed various crystallization method's for the determination of rotenone in derris and described other means of evaluation. The ether-extraction method of Koolhaas (78) was said to be too long. They considered the Jones (62) carbon tetrachlorir^e method to be good only for rotenone contents above 4 percent. The method of Takei, Miyajima, and Cno ( 118 ) was said to be unobjectionable theoretically but ver^- troublesome and time-consuming. The method of Danckwortt and Budde (24) was also mentioned. G-stirner (51) in 1934 compared results by the polarimetric method with those by the ether-extraction and crystallization method. The much lower results by the latter method were explained as largely due to part of the rotenone remaining; in t'^.e mother liquor. Cahn and Boara (13) in 1935 made an extensive study of the deter^-nina- tion of rotenone in 'Ferris root and resin, nvhen derris resins were dis- solved in carbon tetrachloride to peoarate tne crystalline solvate, the eolubilitv was said to be increased to an unusual extent by the presence of the resin. 'A maximum :"ield of rotenone was obtained when 2 cc. of carbon tetrachloride (saturated v/ith rotenone) was used to dissolve each gram of resin. Room temperature was adopted for cr^'-stallization and fil- tration, as little difference was found between results obtained at 0^ and - 19 - at 18° C. Sxcessivs w^shinp- with carbon tetrachloride saturated with rotenone led to marked decrease in yield, with no increase in purity. In the determination of rotonon$ in derris roots 6 to 17 hours' extrac- tion with carbon tetrachloride was not sufficient to remove all the resin or the rotenone. The authors preferred to use trichloroethylene, which usually gave complete extraction of rotenone in 8 to 12 hour?. The rotenone was determined by evaporating the extraction solvent and crystallizing from carbon tetrachloride saturated with rotenone. When pure rotenone-carhon tetrachloride solvate v;as stirred v/ith 5 parts of alcohol saturated v;ith rotenone, a quantitative yield of pure rotenone vra.s obtained. Tests with fixtures of rotenone and "de-rotenon- ized" resin showed that a quantitative yield of the added rotenone was obtained from such a mixture when kept for 3 hours. It was later found advisable to keep such a mixtiire overnight. The alcohol recovery of the separated solvate from derris-root SJSiUples was said to range usually from S3. 5 to 69 percent and rarely from 80 to 90 percent. Tests of purity by methoxyl content, chlorine content., and optical rotation gave values ranging from 89 to 95 percent on samples on which the alcohol-recovery values ranged from 84.5 to 89 percent. The qijestion as to vmether the purity of the solvate should be taken into consideration wnen a rotenone content was stated was considered by these investigators. The yield of rotenone was said to be lower fnan tho amount actually present, and the amount of rotenone left in tno mother liquor might be greater or less than the 10 to 15 percent impurity in the solvate. The value given was there- fore only a minimum fi^ui'e obtained by determining the yield and the purity of the solvate. wTien an appreciable air.punt of pv.re rotenone v;as added to resins containing no rotenone by ths usual prcced;are, an excess of rotenone over that added was obtained. The excess ranged from 7 to 15 percent of the resin in the samples tested. Eesins of this kind were called "Sumatra- type" b" the authors, and the rotenone obtained from them was terT^ed "hidden" rotenone. The highest yields of hidden rotenone were obtained when 4 gm. of resin and 1 gm. of rotenone were dissolved in 10 cc . of carbon tetrachloride saturated with rotenone and the mixture was kept overnight and then filtered as in the usual procedure. Six samples of "normal" resins, containing appreciable proportions of rotenone, gave no significant amount of excess rotenone when treated in this way. However, a sample of root that had given a rotenone content of only 6.2 percent of the resin (1.5 percent of the root) as determined by the ordinary method gave 17 percent of rotenone in tae resin when treated for hidden rotenone. These authors stated that derris rcot should be dried, prior to extraction, in a vacuum desiccator to not more than 5 percent of moisture. The presence of much moisture was said to retard the rate of extraction and also to cause the development of acidity in chlorinated solvents. Drying at 100° C. caused decomposition and low results for rotenone. The procedure proposed by these authors was as follows: - 20 - A quantity of root euTficient to ^ive 5 to 10 gm. of extract is extracted with trichloroethylene for 8 hours in a Soxhlet apparatus, 'ilie solvent is ch3.nged anc' the extraction continued for 4 hours longer. If the second solution is more than pale yellow, the extraction is repeated with fresh solvent for an additional 4 ho^Jirs!. The combined extract is evaporated until the extract b'^coires thick. A gentla current of air is tnen "blown into the flask v/hile the flask is rotated over a naked flame vintil the odor of solvent is replaced "by the odor of derris resin. The flask is weifihed to determine the approximate weight of resin, and 2 cc. of worn; carbon tetrachloride, satiirated with rotcnone , is added for each ff'ram of resin and the resin dissolved rapidly. The solution is cooled, seeded if necessary, snd kept c\'ernight . It is filtered b?- suction through a tared G-coch crucible containing a disk of filter paper, and the solvate is washed with carbon tetrachloride saturated with rotenone until the filtrate is nearly colorless. The crystalline material is dried to constant weight in air below 50° C. The purity of the solvate is tested by the alcohol-recovery method already described. A iiiethod for deterzr.inin.e hidden rotenone in resins poor in rotenone in- volved th-3 procedure alreadj^ described for thir purpose. The authors con- cluded that the usual carbon tetrachloride method f:ave low results if the rotenone content of tn.e resin was belov/ about 17 percent, was seriously in error if it v.'as below 10 percent, and failad completely for resins of verj' low rotenone content. The determination of rotenone by extraction with carbon tetrachloride was described by Pozzi-3scot (93) in 1935. If the deter:'iination was made at rooru temperature, the sa-nple was extracted for IS to 22 hours. Hot extraction, v;hich was recommended by the author, required only 6 to 8 hours. The rotenone was crystallized as the carbon tetrachloride solvate, separa- ted, and weighed. With certain products it was found preferable to extract with acetone or ether. After the solvent had been evaporated, the residue was extracted v;ith hot carbon tetrachloride and the crystallization con- ducted as before. Later in this year the same investij^ator (94) suggested that extrac- tion with acetone, alconol, ether, or eth,vl acetate considerably saortened th--! time required for tiis operation. The use of a "Shumagawa"^ instead of a Soxhlet extractor v;as qlso said to permit more rapid extraction. The extract, freed of solvent, was treated with hot carbon tetrachloride, and the rotenone crj'stallized from this solvent. r-ow:. an (104) in 1935 described the rethod for determining rotenone in derris and cube roots used at the Colonial Institute of Amsterdam. In condensed forn? it was as follows: Evidently the apparatus of Kumagawa and Suto (81), which is also men- tioned by Guillaume and nerve (53). - 21 - To 50 gm. 01 the plant material 250 cc. of chloroform is added in a 500- cc. "beaker, which is covered with a watch glass and allowed to stand 6 hours, with occasional stir- ring. The chloroform solution is then filtered, and the mass on th'j filter is washed with 50 cc. of chloroform. The filter with the mass on it is replaced in the same "beaker, and 100 cc. of chloroforin is added. The mass is stirred again and allowed to stand overnight. Then the chloroform is filtered through a new filter and the mass washed with another 100 cc. of chloroform in two 50-cc . portions. The chloroform is eva- porated completely from the combined filtrates, the last traces being removed by stirrine the flask carefully while holding it over a naked flame and blowing a current of carbon dioxide through it. The extract freed from chloroform is dissolved in 20 cc. of carbon tetrachloride by boiling londer a reflux condenser for several minutes. Upon cooling rctenone separates as rotenone-carbon tetrachloride solvate. '>fnen crystallisation is retarded, the solut-ion is seeded with a small quantity of solvate. The mass is kept o-'-ernight in an ice chest to ensure comr)lete crystallization. The separated solvate is collected by suction in a tared (fritted) glass crucible, v/ashed with about 15 cc . of carbon t=^trachloride (sat^orated with rotenone) in small portions, and dried overnight to constant weight (in air at room temperature). In another article in the san^e year Rowaan (105) compared results obtained on authentic derris samples by a modification of Hoark' s method ( 100 ) and by the polarimetric method of Danckwortt and his coworkers (25). He recommended the extraction methods of P.oark or of Jones (62) or modifi- cations of them as the most workable for the determination of rotanone. In the course of a study by Tattersfield and I.'artin (120) in 19.35 of tae evaluation of derris root, deterninations of rotenone were made on the same samples both in Lialaya, the sc-irce of the material, and at Hothamsted , England. In Malaya rotenone was determined by the method of Oaorgi and Teik (34). At P-othamsted a 50-gm. sample was extracted v/ith ether and the extract was freed of solvent and dissolved in carbon tetra- chloride; the crude solvate obtained v/as recrystallized from alcohol. E-otanone was also determined by th'^ trichloroethylene method of Cahn and Boam (13). In one say.iple an extraction was also made v/ith chloroform. The values obtained for rotenone by these various methods v/ere in general in good agreement. The rotenone content of the samples ranged from about 2 to about 10 percent. In working with the method of Takei , Kiya.jima, and Ono (118) the vn^iters found that in some samples much less rotenone could b'=^ crystallized out from ether by th^ preliminary crystallization recomp^ended by Takei and his coworkers than was separated by the Jones (62) method from carbon tetrachloride. In further work on t^.e evaluation of rotenone-containing plants, Martin and Tattersfield (84) in 1936 studied the effect of removing the toxicarol upon tie separation of rotenone from carbon tetrachloride solu- tions of Sumatra- tyiDe and Derris malacoansie resins. It was found that. - 22 - with a S-j2natra-t;rpe root from which no rotenone separated "by the norm-al procedure, after most of the toxicarol had "been reir.oved "by alkali treat- ment of the ether extract the rotenone separated readily when the residual resin v/as taken up in .carbon tetrachloride. Tv/o Suaatra-type roots frctn waich no rotenone could he ohtainad "by the usual nethod (^/ithout removal of toxicarcl) , ga\'« ahout 2 percent of crude rotencne "by Cahn and Boan^ s (15) procediire for "hidden" rotenone (adding excess rotenone). Purifi- cation of the crude solvate resulted in the p:reatly reduced values of 0.54 percent and 0.67 percent. Aliquots of resin fro^i the same two roots v/ere dissolved in ether, extracted with 5-percent potassium hydroxide, and the ether was recovered from the alkali-insoluhle portion. The residual resin was dissolved in carhon tetrachloride and the solvate crystallised in the usual way. Values of 0.47 and 0.89 percent, respec- tively, of crude rotencne v;ere obtained ty this method, and these results were reduced to 0.40 and 0.66 percent, respectively, hy purification. It thus appeared that in Simatra-type roots the presence of large amounts of resinous material rich in toxicarol prevented the separa-tion of the rotenone present, or treatment with potash removed some other inhihitor of crystallisation, furthermore, the solvate, v>rhich separated readily after treatment with alkali, wa.s obtained in an s.mc^ant agreeing closely with the figure obtained for the purified rotenone by the ncrnal method. With I'er ris malaccer^ sis root, although rotenone separated without the use of t'le lidden-rotenone technique, the procuct was very impiure. Here again t'^^e alkali treatment gave a value for crude rotercne agreeing closely ;\'it'-' that obtained for purified rotenone by the ui'-^al me thod . The authors adciitted the possibility that the alkali carced sorre loss by inducir-A" ozioa'cion of the. rotenone , but they suggested thi.t- an slkali pretreatn:ant such as they described, if suitably controlled, might form the basis of a standard method of rotenone cietern-ination. In purifying the crude solvate by trituration v;ith alcohol these in-'-estigators preferred to filter at 0° C, usin^ alcohol saturated with rotenone at this tempera- ture . Peach (3) in 1936 proposed a serviceable cnethod for determining rotsnon'=i b')Ged on extraction v/ith chloroform, evaporation of an aliquot of the zi\-:^red. extract., and crystallization of the rotenone from ca.rbon tetracb-loride. The method was essentially-- as follov;s: Shake 30 gm. of root with 300 cc. of chloroform in a stop-nered 600-cc. fl^sk at rooir temperature for 2 or 3 hourt. Let stand overnight and then shake 1 hour more. Chill flask and filter contents rapidly into a suitable flask, observing precautions to prevent loss from evaporation. Ad- just the temperature of the filtrate to that of .the original chloroform and transfer a 200-cc. aliquot to a 500-cc. flask. Remove the chloroform by vacuiJir". distillation, and treat with two successive portions of carbon tetrachloride, evaporating under vacuum eacn time to remove traces of -chloroform. Dissolve the extract in 15 to 20 cc. of carbon tetrachloride saturated with rotenon^^ solvate R.t refrigerator temperat\n'e. Induce crystallieatio!: of the extract in an ice bath and allow to crystallize overnight in the refrigerator. Cool the extract in an ice bath, filter through a G-cocn cr^icible as in other methods, and vaoh with carbon tetrachloride saturated with rotenone soDvat^ at room teiipf^ratare . Leave the crucitle on th-^ vacuum for 15 to 20 minutes and then wei^h. Warm (not over 50° C.), a^ain apply vacuum, and reweigh. Repeat this procesr, luitil constant v/oi^ht is attained. Fine grinding of the samplG v/as said to "be ■ot.recesaary in this method; the root need "be ground only to 20 inesh. It was stated tnat the method gave complete extraction of roots of high rotencne content. In 1936 Hobiixson (lOl) prcpo^^ed a method for estimating rotenone in haiaris from ioritish G-uianr,, which involved extraction with carbon tetrachloride in a Soxhlat for 12 to 24 hours, crvstallizeticn of the concentrated and filtered extract for about 22 hoiirs in a freezing chamber, and filtraticsn of the extract after chilling to -10^ C. The crystals were washed with carbon tetrachloride at -10° C., dried in a desiccator over- night, and weighed. Georgi and Teik (35) in 193o urged t^e adcT)tion of a star.dard method for the estimation of rotenone and ,m'^''e details of the method tentatively adopted in the Department of Agriculture of ths Straits Settlements and federated llals-y States. The method v/ap based or one that they had described earlier (34), the chief difference b^ing that recrystaliization of the crude complex from boiling alcohol wag replaced b-/- tritiuation with cold alcohol. Other modif icatioii*- were as follovs: (l) -■ran 20 to 50 gm. of root was used, depending en th'^ rotenone content, ro that not more than 4 gm. of solvate v/ould be obtained. (2) I'he extraction time was reduced to 16 hours by using two b-hour periods and removing the sample and lightly grinding and miring between these periods. The metiiod was said to be satis- factory with all species of Derr is in which th^ rotenone content exceeds 15 percent of the extract. With '^. i^r-.l accer.s is, 'linta type, (Sumatra type of Cahn and Eoam), in which tne rotenone content is about 2 percent , it was necessary to add sufficient rot'?non3 to raise the proportion of that sub- stance to about 30 percent ci the total extract to induce crystallization. It was suggested that tne final figure uight be low because of rotenone remaining in tne mother liquor and son^e passing into soiution when the solvate was triturated with alcohol. Trie possibility of using the weight of the crude solvate and apolying an aporopriate co"^rection factor was sttidied, but the purity of the solvate ■''•aried too widely, both in roots of the same and different species. Consequently tn^? authors did not recommend the weignt of the solvate as n. standard on whicn to base the rotenone content . Buckley (ll) in 193S, in r. stud,;,' of the conc-tituents of derris root, mentioned that long heating of extracts to expel residual solvent v.-as to be avoided, as some change occurred which rendered subsequent crystalliza- tion of rotenone incomplete. In this year also V/orsley (130) msde a study of the deteniiination of rotenone in derris root and the barh of M-ju:idulea sube rcsa . He described ail apparatus for extracting the root by percolation v;itn hot ethyl scetate, wnich comprised a percolator vdth a v/ater .jacket for maintaining an elevated temperat-ure and a receiver immersed in cold v/ater. With this apparatus - 24 - a 10-gm. sample of hif:h rotenone content could be completely extracted v/ith 200 cc. of hot ethyl acetate in about 45 ininates. Hooto of lov/er content, of which larger samples were used, required more solvent anri longer time but seldoM over 2 1/2 nours. Worsley found it advisable to add s^officient rotenone before crystallisation from carbon tetrachloride to bring tne ratio of rotenone to total extract up to 40 percent. The p-iority of tne carbon tetrachloride solvate prepared by the author's method was stated to range from 92.5 to 97 percent and averaged 94.7 percent , as .-"ud^ed by the alco:aol recovery of Catm and Boam. The rotenone obtained ^vfter the alcohol treatment was found by measurements of its optical rota- tion to range in purity from 98 to 99.9 percent, v;ith an average of 99.2 percent. On the basis of the latter value the a.verage purity of the crude solvate became 94 oercent. Worsley pi oposed usin^: this value in calculating the pure-rotenone content of s.. sample, unless particular accuracy v;as re- auired. 'tie also suggested the. use of optical rotation to determine the purity of the solvate, stating that the carbon tetracaloride present had no effect on the rotation. Results by this method v;cre slightly higher than by alcohol treatment. The amo-unt of rotenone left uncrystallized in the resin was not great, as saown by cooling and by adding further large amounts of rotenone and crystallising. Addition of 5 percent of cnarcoal to Derris root before extraction gave a lighter colored extract and a slight but definite increase in the purity of the solvate. Similar results were obtained when 10 percent of chercoal v;as added to i'undulea bark before extraction. The following is a condensed description of the method pro- posed by V/orsley: Sufficient air-dried, ground material is taken to give about 1 gm. of rotenone, and 5 percent by weight of decolori2ing charcoal is added for Derr is or 10 percent for Mundulea. The mixture is run into the percolation tube, which is placed in the constant-tempera- txire bath, maintained at a few degrees below the boJling Doint of ethyl acetate, and the lower end is fitted to a 'liter flask. Suction is applied by means of a water pump. The cs.lculated amount of ethyl acetate is tnen neated almost to boiling mid is poured into tne top of the percolation tube. The smount required is approximately 20 cc. per gram when 10 \.o 20 gm. of material are used, 15 cc. per gram for 25 to 40 gr: . ., 500 cc. for 50 gm., and 600 cc. for 100 {m. of material. Ay -joon as ta^^^ solvent appears at the bottom of t^.e tube, suction is adjusted so that the rate of percolation is about 2 drops per second; when slightly more tnan half the solvent has come through, it is inci'eased to about 4 drops a second; and finally, when practically a.li the solvent is througn, full suction is applied. The extract is filtered into a distillin.?- flasi" ar^d prac- tically all the eth:/^l acetate distilled off. The resins are transferred to a small, v/eighed leaker, and the ethyl acetate is removed on tne water catn. The beaker is weighed to deter'nine the amount of resins. Sufficient ourifieri rotenone (40-mesn) is then added to bring th^ rotenone content in the resins up to at least 40 oercent; in any case at least 1.0 gm. is added. It is stirred into the heated resins on a water bath, 2 cc. of - 25 - carbon tetrachloride satiorated with rotenone for every cram of' r=»slns plus rctenone is added, and the mixture is warmed until solution is complete. The beaker is set aside until morning ' in a desiccator containing a dish of carbon tetrachloride; seeding is unnecessary. The crystals sre filtered through a Gooch crucible on a disk of filter paper. They are washed with solvent (carbon tetrachloride-rotenone) until no further color is removed and dried for about 6 nours at about 40^^ C. The weight obtained tiacc 0.719 gives the amo-ont of crude rotenone. The purity is most accurately doterminsd by triturating the rotenone-carbon tetrachloride complex with absolute alcohol saturated v/itn rotenone, 5 cc. for every grrjn of complex, and leaving overnignt in a c'.esiccatcr containing a cish oi alcohol. The rotenone is collected iu a Gooch crucible, washed with 30 to 40 cc. of the solvent, and dried at 100° for 6 hours. Frpm the wei£:nt of rotenone thus obtained the amount originally added is deducted; tie difference is the amount in the sample. The purity of this rotenone may be deterrnined by weighing out between 0.48 and 0.50 gm., dissolving in a stoppered vessel in 10.00 cc. of pure benzene, and ceteri^iining the angle of rota- tion in a 200-mm. tube. From a previously prepared curve, or from the formula C = (alpha - i.428)/4.066, tne concentration of pure rotenone is obtained and the purity thus determined. The . purity ma^/ be taken as bein^; 95.2 Toercent, and this means tiiat for rotenone contents above 6 percent a correction of -0.1 percent is made, but for contents below 6 percent no correction is necessary. A less accurate result for pure rotenone can be obtained by determining the angle of rotation of the rotenone-carbon tetrach- loride complex by weit;hing out betv/eeh 5.8 and 7.2 gm. arid dissolv- ing in 10.00 cc. of pure benzene. As before, tne concentration of pure rotenone is determined and the percentage purity of rotenone in the "complex calculated; from this figure 2.6 is deducted and the difference used to calculate pure rotenone in the sample. An even more rapid method is to assume taa purity of the comr- plex to be 94 percent and to calculate r)ure rotenone on this basis. Sxcept for a few unusual derris samples results of sufficient accuracy for routine estimations are obtained. In 1936 many comiaercial analysts of derris and cube roots were using a method for rotenone developed by H. A. Seil.^ This method has not been publisned, and several variations have been brought to the attention of the reviewer. In this method 50 gm. of root was extracted with carbon tetrachloride in a Soxhlet for 5 to 7 hours, or longer if necessary. Before extraction 0.34 gm. of rotenone-carbon tetracnloride solvate v/as added to the extraction flask, and after extraction the extract v/as concentrated to 40 cc. Crystallization and filtration of the solvate were carried out as c Details of this method were obtained in 1936 in private communications from licCormick pjad Co. and John Powell and Co. - 26 - in other methods. The crystals were washed with a cold saturated solution of rotenone in carton tetrachloride and allowed to air-dry to constant weight. The value for rotenone was calculated from this weight of crude solvate . Eowaan ( 106 ) in 1936 again warned against the use of Danckwortt's (85) pclarimetric method for the determination of rotenone and stated that the most reliable procedvire was some modification of the ex tract ion- crj.'stal- li7ation method. The appearance of microscopic cr7,'-stals of rotenone was descrited "by Poezi-ilscot (96) in 1936 as an aid in the identification of this mater- ial in analytical work. In 1936 government agencies of the Dutch Sast Indies preoared a sample of derris root and sent subsacples for analysis to nine labora- tories in various parts of the world. In 1937 Koolhaas and J'eijer re- ported the results of this investigation. Values for rotenone on a mois- ture-free basis were as follows: "P.otenone Laboratory Percent A — United States Department of Agriculture (Jones) 8.1 (pure) B — Department of Agriculture, btraits Settlements and federated r.alay States (Georgi) 6.6 (p\ire) C— Commercial Museum of the Colonial Institute, Amsterdam (Howaan) 9.7 (crude) D — The Cooper Technical Bureau, London (Csim and Eoam) 7.7 (pure) 3 — Rothamsted Experimental Station (Tattersf ield and Martin) 8.4 (pure) F— Seil. Putt, and Rusby, New York (Seil) 7.5 (crude) G — Caeser and Lorentz, Kalle-Saale , Germany 12.3 (crude) H — Diethelm, Ltd., Singapore 6.3 (crude) I — Laboratory for Chemical Research, Buitenzorg, Java (Koolhaas and .''eijer) 9.8 (pure) See footnote 1, - 27 - Various methods of extraction wera used, but in all except tv/o of the laboratories the rotenone v/as separated as the carbon tetrachloride solvate. Laboratory A used both Soxtilet extiaction and maceration with refluxin^; the latter method, using benzene, was preferred; the crude solvate was p-orified by an alcohol-recovery test. In laboratory B the sample was mixed with ssjid and extracted with carbon tetrachloride for tv/o 6-hour periods; an alcohol-recovery test was included. The method used by laboratory C involved maceration v/ith chloroform for two extended periods; no test of the purity of the solvate was made. Laboratory I> made comparative Soxhl,-«t extractions with trichloroethylene , chloroform, ana ether; ether extrac- tion gave markedly lov;er values, and the first metnod. v/as preferred; alcohol recovery v/as used for purification. Sther extraction of the sampl? mixed with sand in a Soxhlet for 24 hours v/as used by laboratory 3; the usual alcohol-recovery test was employed. Laboratory F extracted the sample for 6 hours in a Soxhlet with carbon tetrachloride and used no final purifica- tion. In laboratory G an extraction method usin.ff chloroform in a Soxhlet was compared with the optic^^l rotation of a benzene extract of the sample; both methods gave results much higher thajn these obtained by other labora- tories. Ether extraction in a Soxhlet for 6 hours, followed by crystal- lization directly from tne ether, with no purity tests, was the method used by laboratory H. The Isiboratory of the authors of the report (labora- tory I) used Soxhlet extraction witn ether for 48 hours, followed by crystallization from the ether and measurement of the purity 'bj determi- nation of the melting point. Koolhaas and !!eijer criticized the results on the basis that many of the laboratories did not obtain complete extraction of the root. Ben- zene, chloroform, and ether, the last-named as used in their laboratory (I), were stated to be suitable extraction solvents, but longer extraction than was used by i/;ost of the laboratories was said to be necessary. Use of melt- ing-point deteridnation in .judging the purity of the separated rotenone gave higher values thaja alcohol recovery, and these p.uthors preferred this method. They also pointed out that the optical rotation of the whole extract is of no value in d.etermining the rotenone content. They recom- mended that for analysis derris root should be ground so that at least 75 percent passes an 60-mesh sieve, and that the moisture content sho\ild not be over 12 percent. Georgi and Teik (?6) in 1937 stated that rotenone may be lost at two stages of its determination. Tiras , some may not crystallize from the carbon tetrachloride but may remain in the mother liquor, while some may pass into solution when the co/nplex is trittirated with alcohol. i3eterjnina- tions of the optical rotations of alcoholic filtrates after recovery of rotenone indicated that the loss in triturating the complex witn alcohol amounted to approximately 6 percent, calculated on the weight of rotenone recovered. It vras not found possible to devise a method for estimating the a-Tiount in the carbon tetrachloride mother liquor, but the low optical rotation of the residual bodies in this liquor pointed to a smaller degree of retention than with the alcohol liquor. Tae total loss was estimated as possibly 10 percent. Begtrup (4) pointed out in 1937 that ej.tr-'.ction of derrls or cute root '.<;ith a low boiling solvent, such as ether in a So:chl9t apTDaratus, is incomplete. On the oth^r hand, he stated that if a r.olvent of higher "boiling point is used tie prolonged neating generally destroys the extrac- tive substances arid t>>us renders crjfitalli nation of th-^ rotenone difficult or impossible. To overccne tcese difficulties h^ reconir-snded extraction with toluene at room temperatiare . The procedure suggested "by Pegtrup was as follov/s: Thirty grsjts of lOO-mesh material is packed in an ordinary funnel, thoroughly rcipter.ed with toluene, and v/ashed si::: times with 20 cc. of toluene (eac.i extract is per-nitted to drain off ccmpl3tely). Tne co-rbined e;:tract is diluted to 150 cc, and a 50-cc. aliquot iste-ken. Tie tolu^ine is distilled off on an oil bath maintained at about 130° C The residue is dissolved in 7-8 cc. of carbon teti-achlcrice saturated with rotenone at 10° C. and transferrsd to a weigrhin^ bottle. Washings bring the total volume to 12-15 cc. Cry p.t alii '/--it ion is ailov/'^d to proceed over- night at 10° C. Tne cryEvbals are filtered, wc-shed, and dried, as in otner methods. Chevalier and Chevalier (39) in 1S?7 described a method involving extraction wit'a cnlorofcrm and crvstallization from carlcn tetrachloride, similar in all essential detai?.s to "Dreviov'-S methods. The rotenone v/as purified by recrystalliz.--tion from warm alccnol. Ripert (99_) used dichlorcethylene for the -extraction of rotenone. The determination of rotenone in samples of I^ erris and Ilundulea was described by Guillaure ??jid Froeschel (5;=) in 1937. They used a modification of tie method of iCoclhaas (TE^) . Extraction of the 5-gra . sample with ether in a continuous percolator was cai-ried out as described in the section on Total Sxtrsct. The evaporated extract was then treated as follows: Add 15 cc. of cold ether to the extract in a tared crystal- lizing dish (A), covej, and let stand in the refrigerator for about 24 hours. The extract dissolveo and the crystals of rotenone deposit. Decant the solvent into a second tared crystallizing dish (B) . Hapidly wash the crystals v/ith a little cold ether. Lecant the wash liquid into the crystal- lizing dish (B) . Keep this dish in refrigerator for 24 hours. Dry crystallizing dish (A) in oven at 50° C. for 1 hour and weigh. If crystals form in dish (3), decant the liquid, wash as before, dry dish and contents at 50° C, weigh and add to weight of rotenone in dish (a). These investigators attempted to check the sensitivity of the crystal- lization method. They added to the powdered root of a species of Lebec^tia that contained no roterone 'oy qualitative testr, progressively increasin^^• amo^onts of rotenone in the form of carbon tetrachloride sol- vate and analyzed the mixtures by the methods indicated. As shown in the following tabulation, very good recovery of the rotenone was obtained: - 29 - HotenoDS added per 5 gm. Hotenone found (as pure rotenone) ("by ether method) • ' ' ■ Grain Gi*am , . • 0.3451 .0713 .0683 .0348 \. ' .0378 They. stated that when less than 0.0355 gm. of rotenone was" employed values w.ere too low. Determinations /by chloroform extraction (5-gm. sample) and crystallization from carton tetrachloride on some of these prepared samples, as well as on samples of Serris and IJundulea, gave results in good agreement v/ith those "by ether. Levallois (82) in 1937 said that ethyl acetate and chloroform were tne two hest extraction solvents for use in, the determin'^.tion of rotenone. He emphasized that all trace of extraction solvent must' ha eliminated under vacuum, before crystallizing from carbon tetrachloride. Levallois stated that . the tendencj^ was to abandon the colorimetric methods in favor of the gravimetric methods for rotenone. • Crystallization methods for rotenone were said by Schonberg ( llO ) to be open to the objection that they were not applicable to very small amounts of rotenone , that the results were not in accord with the in- secticidal activity, and . that they were very laborious. It was stated that the carbon te.trachloride complex contained variable proportions of impurities and ths^t its content of pure rotenone could not be deduced. Colorimetric methods were recommended. ^orsley (l3l) in 1937. stated that prior to the publication of the work of Cahn and Boem,(l3) he had found that addition of pure rotenone to extracts of I.!undulea suberosa bark resulted in a considerably greater net yield of rotenone. He obtained hi.'jher yields of rotenone by adding decolorizing charcoal to the powdered bark before extraction with ether. It was necessary, however, to increase the time of extraction and better extraction solvents were tried. Percolation with hot ethyl acetate gave the most satisfactorv results. Details of this method have been described (130). Eowaan (107^) in 1937 recommended the use of. chloroform at room temperature for the. extraction of rotenone. he stated that drying at 50° G. for 1 hour brought the rotenone-carbon tetrachloride solvate to constant weight. Rovraan tentatively reccramended determination of the purity ,by alcohol recovery, although he said that this process needed im- provement . ., .Heijer (86)>,in! 1937, before a meeting of the Euitenzorg (.Tava) Experiment Station stai'f , .discussed the evaluation of derris and reviewed methods for. the. determination of rotenone. Treatment of the mother liquor from rotenone separation by chromatographic adsorption, by formation of the hydrazine of rotenone, and by other methods, was said to show as much - 30 - additional rotenone as 10 percent of the remaining extract. Of three methods of determining piirity alcohol recovery was said to give the lowest values, the melting-point method the highest, and the polarization method intermediate values. Because of ease of handling the last two were pre- ferred. A method for rotenone usini"- ethj'l acetate for extraction was sug- gested "by Pozzi-ilscot (97) in 1937. Extraction wa"B accomplished "by boiling under refliox. The extract, concentrated to a sirup, was treated with sufficient activated charcoal to make a dry powder and then extracted with a known vclvune of carbon tetrachloride already saturated with rotenone, This extract was diluted with carbon tetrachloride if necessary. The extract was made to the original volume of the carbon tetrachloride solu- tion and set aside to crystallize for 24 hours at the temperature at which the solvent was saturated with rotenone. The crystals were filtered and weighed in the usual way. Seaber (ill) in 1937 reported analyses of derris, barbasco, and timbo for rotenone content using carbon tetrachloride, chloroform, trichloroethylene, and ethyl acetate as extraction solvents. In the case of carbon tetrachloride the srunple was extracted, at least 16 hours, and during crystallization the extract was kept at room temperature for 2 days and then in ice at least 3 hours before filtering. The chloroform extrac- tion method was- essentially that of Beach (3). The error introduced by change in volume due to solution of the extract was found to be small and was shown to be almost exactly compensated for by the effect of evaporation during filtering. Instead of using a mechanical shaker in this method it was found that equally good results vv^re obtained by allowing the sample to stand in chloroform overnight , shaMng occasionally by hand the next day, allowing to stand over another night, and filtering the next day. Extraction in a Soxhlet with chloroform v/as tried, but the solvate obtained was letss nure . The trichloroethylene method of Cahn and Boam (13) and the hot ethyl acetate percolation of Worsley (130.) were also tried. Purity of the solvate was calculated by polarization in benzene, which gave re- sults a lit tie higher than those by the alcohol-recovery method. In almost all cases the chloroform method irave higher results than did carbon tetrachloride, and in some cases the differences were large. The question as to whether these differences were due to failure of the carbon tetrachloride to extract the rotenone or to decomposition was studied. In one typical extraction 62 percent of the rotenone was obtained in the first 8 hours' extraction with carbon tetrachloride. However, with this solvent a limit seemed to be reached before all the rotenone was extracted out of the root. Decomposition of rotenone in boiling carbon tetrachloride was found to be a factor in lower results by this solvent. Boiling rotenone in this solvent for 16 hours gave a loss equivalent to about 0.2 percent on a 5-percent rotenone root, and for 72 hours a loss equivalent to about 0.5 percent on a similar root. It was stated that carbon tetrachloride cannot be relied upon 'to extract rotenone completely. Extraction by the ethyl acetate method gave result^ in agreement with those by the room-temperature chloroform method but the solvate obtained was less p\ire. Seaber recommended as the best method for commercial purposes extrac- tion with cold chloroform, crystallization from carbon tetrachloride,' and determination of purity by polarization, the result to be reported in terms of pure rotenone . - 31 - Seaber jilso investigated the possibility of making "use of the maximmi of the ultraviolet absorption curve to estimate the percentage of rotenone in the crude solvate. Sxtreme dilution was necessary, but never- theless results close to those by polarisation were obtained in some cases, although in others the values were too high. The method could not be used as a routine process for purity but might be useful in identifying and roughly estimating rotenone present in small amounts in mixtures. An attempt to estimate rotenone by this method in whole extracts of the root was not successful because of the effect of imp\irities. Jones (65_) in 1937 studied the crystallization -f the rotenone- carbon tetrachloride solvate from extracts and proposed a modifier! loro- cedure for thif. step in the rotenone determination. The proposed crystal- lisation procedure was as follows: The solvent-free extract from a 25-gii. sample of root is dissolved in 25 cc. of carbon tetrachloride, cooled in an ice bath, and seeded with crystals of rotenone-carbon- tetrachloride solvate. If only a small amount of crystalline m-.terial sepa- rates, an accurately weighed, amount of rotenone is add.ed, so that at least 1 gm. of pure rotenone is present. This extract and a wash solution having 0.27 gm. of rotenone for 100 cc. of carbon tetrachloride are maintained at 0° C. in an ice bath overnight. The crystals are then filtered in a tared Gooch crucible, washed with 6-10 cc. of ice-cold wash solution, and dried to constant weight at 40'^ C. One gram is treated with 10 cc. of alcohol saturated witn rotenone at room tem.perature and set aside at this temperature for 4 hours. This material is filtered throu^gh a tared C-oocn crucible, washed with about 5 cc. of alcohol saturated with rotenone at the same tempera- ture, and dried to constant weight at 105°. Corrections are made of 0.07 gm . for the rotenone dissolved by the 25 cc. of carbon tetrachloride used and also for any rotenone added. The purity of the solvate was found to deoend principally upon the propor- tion of rotenone to total extract and upon the proportion of solvent used in crystallization. As the so-called ".pure" rotenone obtained "oy the alconol treatment was' not entirely pure, and as its purity depended on the purity of the solvate from which it was prepared, it was desirable to obtain tne solvate in as nearly piare a form as possible. lor this reason the author preferred to crystallize the solvate from a larger proportion of solvent than that used by Cahn and Boam (l3) . '.'ore rapid conversion of the solvate to rotenone was obtained when 10 cc. of alcohol per frarn of solvate v;as used rather than the 5 cc. proposed by Cahn and Boam. A determination of the precision of replicate results on one sample of derris of about 4-percent rotenone content showed a standard deviation of i 0.05 percent. A' study was ma.de of trie accuracy of the proposed crystallization procedure when applied to extracts of both derris and cube roots v;ith various proportions of rotenone to total extract. The method used assumed that the ixonrotenone portion of the extract exerts only a retarding effect on the crystallization and has little or no actual sol- vent effect on the rotenone, an assumotion tnat was indicated by all the - .?2 -■ writer's v/ork up. tc this, point. On thin assur.pti-on extracts of approxi- mately knc'nT^i rotenone contents were prepared from large samples' of roots of 4-percent rotenone content or over. The extracts were subjected to a preliminary crystallization in the usual way, and the solvate was removed hy suction filtration. The filtrate was then made to a definite volume, and aliquots were taken of such a si-^ie as to he equivalent to 25 gm. of root samples. The amount of rotenOne remaining in each aliquot wa.s calculated from known soluhility figures. To the dried aliquots amounts of pure rotenone varying from 0.2 to 2.0 gm. were added, each was treated with 25 cc. of carbon tatrachloride , and crystallization was carried out "by the proposed method. The actual weij^ht of pure rotenone obtained was used witnout correcting for solubility in the carbon tetrachloride. Hence from the knov.n solubility of rotenone in carbon tetrachloride at C^ C, a loss of 0.07 gra. was to be expected. ' The values for rotenone present (amount calculated from solubility plus anount added) were plotted against the amount lost in crystallisation (amount present minus amount actually recovered). In extracts with norrcal proportions of extractives other than rotenon? , the Iocs of rotenone in crysoalli-jation wfs great when only small amounts v/ere present, but decreased with increasing amounts of rotenone until at O.Oo to 1.0 gm. it he came pr^.ctically constant at approximately the loss to be expected from the solvent effect of 25 cc. of carbon tetrachloride. Similar results wore ohtained with extracts of the same roots preriared to hs.ve both a.bnorina-liy 'ligh and abnoi-mally low proportions of nonrotenone resins. Alien extra,cts v/ith normal proportions of rotenone were allov/ed to crystalline for 48 hours, coirplete crystal- lization to a value approximately equal tq the solubility was obtained v*ien only ahout 0.6 gm. of rotenone v^as present. An extract of a Siir.atra-tyoe derris root was subjected to the same preliminary crystallization except that sufficient pure rotenone was added to assure quantitative crystallization. The filtrate was treated in the same way as described for those of the 4-percent roots. When about 1 gm. of rotenone was present, the loss on crystallization was constant and approximately equal to the solubility loss; consequently these resins are similar to ordinary resins in tneir effect on rotenone crystallization. The author therefore believed that the "hidden" rotenone described by Cahn and Boam (13) was a result of the retarded crystallization obtained with any extract having a low proportion of rotenone to nonrotenone resins; the addition of rotenone merely hastened the crystalliaation. The accuracy of the Cahn and Boam method, in which 1 f^a. of extract is dissolved in only 2 cc. of carbon tetrachloride, was briefly studied and appeared to be equal to that of the author's method. It v/as concluded that accurate results by the proposed crystalliza- tion method were obtained only wnen the rotenone present was equ.ivalent to 4 percent of the root, or waen sufficient rotenone was added to bring the amount present during crystallization above this value. lor the extracts studied the method' gave values which, in view of the precision, were not significantly different from the actual rotenone contents. Because of tae widely varying composition of different samples of derris and cube root, no general estimate was made of tn? accuracy. - 3S •- In 1938 Jonea and • Graham (71,')- f5tu(?i?d,, methods .for .t.he q-aantitative extraction cf rotPnone from dsrris and cub© roots. The following:; general methods v/ere ccmpcred: (1) Soxhlet extraction \ir.:.n?r car-tori tetrachloride. (2) ioillng-rultip? e extraction:, in v/hich the sample waa refliijced vvith t'-^e co Lrent on the c-te:?in '£,ath and theu filtered "by Eviction ax.d the jn^vc vas tr&fitef", tvdce with fresh solvent in the S'-^jne \\a'r . Benzene, carbon tetrachloride, chlcroforti, ethylene dichloride, trichloroeth'^lsne , ethyl acetate, and the berzene-sloohol azeotropic mi-.rture v/ere tested as solvents by this method. ■ :,;■ • iZ) Boiling-aliquot nethod , • in vhich the , sariple was treated vdth a weighed amo'int cf solvent, refiv-xed on the strain bath, cooled, riolvent added to-,rer>lace that Irst, the extract filtert^sd, en J. an aliquot taken. Only benre^.e was tried in this :.ethod; (4) Rooni temp-jratiire-ri.ulti^^le e:?tisction method, similar to method (2) but carried out at roo;n temperatirj-e. Only chloroform was tried'. (s) Hoom temperature-f^liouot mrtn'-':, ruDstantia] ly the Bome as that prooos'-^d bv Bes^a (-5). . Chlorof orrp , beii^ene, and ethyl acetate v/ere tested. After r^mova: of the solvent, cr^-?,talli'.'ation of the rotenone was carried out by the r.ethod proposed by Jones (6o). The marcs from multiple- extraction niethods were tested for rotenone by a qualitative color test. Tests were r^ade o.^ a la: p-e nujn^er of finely powdored samples of derris, cube, and timco roots az-A on one samplo of Tophrosia virginiana root. Marc<3 from toe ben73ne-ooilin.ff-i-u.ltiple extraction method and tne chloroform-room temperature-mult i"ole extraction method shov/ed practically complete extraction of the rotoi.crie . Eesults for rotenone by these methods were in agreement with f'iOse by the chloroform-room temperature- aliquot method. The latter method was preforred b-'- the authors because of its convenience. Various phases of the chloroform-room temperature- aliquot method were t-ien studied. It v/.as found that the time of shaking during extraction mig:ht be; reduced, to -2 ho^JLrs without seriously; affecting the results, but to ensure ■ complete extraction overnight shaking v/as advised. Firmness of th-^ sojnole vas an important factor in obtaining complete extraction. Results or. co-arcely ground ramplen wore in some cases 1 riercent lov/er than on th3 sajne.roptE regxound to a finer size. It v;as stated that, to give satisfactory extraction by the aliquoting procedure, coarse samples should be gro'jnd so that at least 95 percent passed a 60-mesh sieve. Samples containing a high ratio of rotenone to total extract were found to be .'nore difficult to extract than tnose with lower percentages of rotenone. Wneoi the ratio of rotenone to total extract was about 40 percent or over, particularly in the case of derris roots, it was necessary to employ the chloroform-room temperature-multiple - 34 - extraction method to obtain s&tiafactorj.' extraction. Cube roots in general were more readily extracted of their rotenone content than were derris roots, Preliminary drying was unnecessary, since tne r.oisture content of rerris and cube roots as received in the United States was found not to be suf- ficiently great to interfere wlfi their analysis. In addition the results for rotenone and the purity of the solvate were lower when the r^ot was dried either at 100° C. or at 50^ under vacuum before analysis. In the chloroform-room temperature-aliquot method replicate results on a sample by a single investigator in general agreed within about 5 percent. The average difference between the results by the two authors was only about 3.5 percent. As a result of this work Jones and G-raham (72) in 1938 proposed a complete extraction and crystallization method for rotenone based on the room temperature-aliquot extraction using chloroform and the crystallisa- tion as proposed by Jones. The method with some modification has recently been adopted by the Association of Official Agricultural Chemista (49) and is described in detail later in this section. For roots of abnormally high ratio of rotenone to total extract, or in any case of doubt as to the completeness of extraction, the alternative room-temperature extraction with successive lots of chloroform was suggested. Cahn, Phipers, ana Boam (l7) in 1938 r'iscussed methods for deter- mining the various constituents of derris extract. They did not agree with Jones (65) that the nonrotenone resins exert no solvent effect on the rotenone. They cited as experimental evidence one derris extract which gave 39 percent of rotenone by the usual method including correction for purity, but gave 42 percent of pure rotenone when a first crop was crystal- lized and the mother liquor was allov;ed to be adsorbed on charcoal and rotenone recovered from the numerous fractions obtained. Hence the authors believed that the rotenone content calculated from the crude solvate W8.s, by a compensation of errors, closer to the correct valu^:^ than results based on pure rotenone. They tested the effect of the removal of the alkali-soluble material as suggested by I'artin and Tattersfield (84) , and found that in a series of Sumatra-type extracts substazitially the same results were obtained by this method as by the nidden rotenone tech- nique of Cahn and Beam (ij?) in which excess rotenone is added. Thev also discassed results by the Soodhue modification of the Gross and Smith color test (40), which determines primarily rotenone plus deguelin (see section on Def^elin and Rotenone plus Deguelin). They found that witti the numerous samples of derris extract studied (except Sumatra-type extracts) the fol- lowing;, relation held: Goodhue value = percent rotenone + 22 ± 3, v/hara the values are expressed as percentage of the extract and the rotenone value is that for crude rotenone. They stated that this relation had been used successfully in the inverse sense to deterinine approximate rotenone con- tents from Goodhue values, and was especially valuable for this purpose when only small amounts of material were available. Rowaan and Van Duuren (108) in 1938 recommended room-temp<»Tature extraction with chloroform and removal of an aliquot for the detarmination of rotenone in Derri s and Lonchocarpus roots. - ?5 - Seaber (ll_2_) in' 1938 putlifhe.^ results' of analyses of derris, timbo, barbasco, and cubs 'roots by the room tempdratiire-chloroform-aliquot extrac- tion method of Beach (3) and the short-time (6 hours) carbon tetrachloride Soxhlet extraction of S3il (see p. 25). The first method almost invariably gave higher results for (""erris and generally lower results for timbo and barbasco, while results for cube v;6re only a little hi^^her by the first method. It was suggostec' tliat derris roots be a-jsayed by the room tempera- ture-chloroform method, v;hile other roots be analyzed by both methods and the higher result taken. As in previous work, Seaber determined the piiri ty of th'3 solvate by polarisation. The following method for the isolation of small quantities of rotenone from oleaf^lnous seeds v;as described oy G-ui chard (52) in 1938: TTie saraole is extracted in the cold wif": ether or petrolerjn ether. After evaporation of the solvent the oily liquid is extracted with 50-percent aceti'3 acid in a separatory funnel until the last lot £:ives no color reaction for rotenone. This acid solution is extracted with ether and t-is 3th?r evaporated. The residue is dissolved in aboj.t 10 oc. of 50-percent acetic acid, and the solution is filt'3:.'6d end dried under vacuum until all the acetic acid is removed.. Tnis :"esidu;? is dissolved in several cubic centimeters of ether and placed in a partly covered weighing bottle in the refrigerator for 2 v.-eeks to cryptallire. If crystalli7ation does not occur in this tine, tho solution is evaporated and the residue, dissolved in 4 to 5 cc of 50-percent acetic acid, is filtered and dried as before. This residue is now dissolved in 2 to 3 cc . of anhydrous ether and left in the refrigera^tor as' before for 2 weeks, wlien crystallisation has occurred the ether solution is decanted, the crystals are dissolved in cold anhydrous ether, and this solution is replaced for crystalli-^ation as before. By such successive crystallisa- tions as this pure rotenone is obtained. The determination of rotenone was discussed by Koolhaas and I.'eijer (79) in 1938. Not only was some rotenone left uncrystallized but varying amounts remained in the mother liquor. As an extreme case, that of a root with a total-extract content of 25 percent a,nd an a>.ctual rotenone content of 3 percent, rotenone amountini-r to 2.2 percent might be left in the mother liquor. In a root of this type by the usual method only about 60 percent of the true rotenone content would be obtained. AJethods for determining rotenone were discussed at length by Bertaud-E-ossi (6) in 1938. The method of Jones (o^) was said to give satisfactory results, but several changes designed to give more rapid results were introduced. To obtain ready crystallization of the rotenone from carbon tetracnloride , the extract should be dry. Iiather than dry the sample itself end thus ca.use decomposition of the rotenone, this in- vestigator dried the carbon tetrachloride extract over anhydrous sodium sulfate. The time of crystallization varied with the proportion of rotenone to resin; crystallization took place rapidly in extracts rich in- resin. A device for enclosing the filtering crucible in an ice bath to maintain the - 36 - filtration at 0° C was described ano illustrated. In case a rapid result was desired, instead of waiting for the carbon tetrachloride solvate to come to constant weight, it could be dissolved in ether, the solution evaporated, and the process repeated. This scheme sufficed to remove the carbon tetrachloricie completely, and the dried product was weighed as rotenone . Mei.jer (67) reported in 1958 that preliminary drying of the derris root sample at 50° and 80° C. caused a marked lowering in the rotenone results. Thus, a sample originally analyzing 10.6 percent of rotenone gave the following results after drying: TeraiDerature Time Eotenone '° C. Hours Percent 40 2 10.3 60 2 6.1 80 0.5 6.2 80 2 5.2 The Imperial Institute (58_) reported in 1938 on work done by the Department of Agriculture of the Federated I'alay States. Soxhlet extrac- tion with carbon tetrachloride had been abandoned, and extraction with cold chloroform had been adopted as a routine method for rotenone. Results by this method fell only a little short of those by extraction with hot chloroform and were in agreement v/ith results obtained with hot ethylene dichloride and ethyl acetate. Graham (44) , in his 1937 report to the Association of Official Agri- cultural Chemists, described work on th^ determination of rotenone done in collaboration with Jones, and recommended that the chloroform extrac- tion method of Jones and G-raham (72) be adopted by the Association as a tentative method. A titrimetric step in the procediire for determining rotenone, mak- ing use of the rotenone-dichloroacetic acid solvate, was described by Jones (o?) in 1938. With dichloroacetic acid rotenone was found to form a solvate containing an equimolecular proportion of the two constituents. Formation of this solvate v/as adapted to determining the purity of the crude carbon tetrachloride solvate obtained in the usual gravimetric crystallization method. The method v?as briefly as follows: The carbon tetrachloride solvate obtained in the usual way is dissolved in acetone, and the solution is evaporated to remove carbon tetrachloride. The residue is dissolved by warming in 10 cc. of 60-percent dichloroacetic acid. Tne solution is cooled in an ice bath, 10 cc. of water is added slowly, and crystals of the dichloroacetic acid solvate are added for soeding. After the solu- tion h^s been 2 or 3 jf.inutas in the ice bath, water is added 10 to 15 drops at a time, with cooling in the ice bath between ad.di- tions, until about 25 cc. has been added. Then 25 cc. more of water is added dropwise and finally, after further cooling, 50 cc. of water is added more rapidly. After some additional cooling the crystals are filtered on a Gooch and washed with about 250 cc. of - 7^7 - wate-r. The crystals are then dissolved in chloroform', atout 50 cc. .of vater is added, and the. mixture titrated with O.I.N alkali usinf phsnolphthaloin ?"-s inc'lcat-or . With proper care in th? acicUtion of v/ater during the precipitation, the acid solvate separated in a crystalline form from which excess acid was readily washed. In tests on specially prepared cart.cn tetrachloride solvates, the method ^ave values for purity about 2 percent higher than those "by the older alcohol-recovery test. Results on samples of pov/dered root were in good agreement with those by the older ^avimetric procedure. The procedure effected a saving in time over the .-Tavimetric method and neutral impurities, such as sulfur, did not interfere. Attempts to pre- cipitate the acid solvate directly from whole derris and cubs extracts in an effort to shorten th-^ procodure still further met v;ith failure. In his 1938 report to th--- Ass<^ciation of Cfficia.l Agricult-or-al Chemists, Graham (47) r?v? the results of a collaborati''-e study comparing the crystalli?:3tion method of Jones and G-rah.am (72) with the titration method (S7). The former method .frave good results on both derris and cube, but several collaborators had difficulty with the latter procedure. At the 1933 meeting of the ilational Association of Insecticide and disinfectant i-anufact"'arers, Graham (45) reviev/ed methods of analysis for rotenone. Extraction of the sample with carbon tetrachloride in a Soxhlet, although widely used, was said to have several disadvantages, amon^r which were difficulty in obtaining complete extraction and decomposition of the solvent due to moisture present, with consequent decomposition of the rotenone. Chloroform extraction at room temperature was recommended. It v/as stated that a modification of the crystallization method na,d been developed for the dete^rmination of rotenone in the presence of sulfur. The method was not described, but it ivas said thjat the rotenone a,nd con- taminating sulfur v/ere weighed and a correction was then made for the amount of sulfur present. Tae- titration method v/as also said to be applicable to such mixtui-es. Details of th? rr.-^tho'' used in the pr-^sence of sulfur have been furnished by the Agricultural Marketing Se'-vice ( 126 ) . Extraction v/as carrif rotonone in th^^ solvent v/hile at the other an excess of crystals was kept iij the sol\-ent in the refrigerator. The former method was now agreed to bf^ the bettor. SoTce analyses were made by the two general methods and using titra- tion to determine purity (c7) . In method I abnormally high results were obtained on the Kinta-typa root, but in method II, a.fter removal of alkali- solubla material, this root gave values that agreed with previous figures by. other methods of measuring purity. The titration method appf»ared to be applicable only to solvates of a certain degree of p-urity. In 1939 C^^hn sjid Boem (l6) confirmed the finding of Jones that the dichloroacetic acid and 'alcohol-recovery procedures gave substantially the same results. The relation between rotenone content and values by the Goodhue (40) color' test was studied fui'ther. There was found to be an approximate relation between the values by this test end. the pure-rotenone content, but the correl'ition was more exact when crude-rctenone content w?*s .considered. , An abnon-ally small difference between Goodhue value and rctenone content was said tc indicate the presence of Sumatra-type root- or extract. ■ In 1939 GrahajTi (46) reported that, in the an.alysis of cube powders by the chloroform-extraction method, higher percentages of rotenone were obtained, and the rctenone-carbon tetrachloride solvate crystallized more readily Piid was purer v/aen decolorising carbon was used in the extraction flask, \vnen 10 gsi. (Forit-A)' was mixed with 30 gm. of cube powder and extracted in the usual way, the values for rotenone were from 0.5 to 0.8 percent higher than when no carbon was used^ The use of carbon with the derris tested caused no significant difference in the results for rotenone. Braak (9) in 1939 reviewed the resxilts of analysis of the sample of derris root submitted by the Dutch 5ast Indies authorities to various laboratories throughout the v;orld and which previously had been reported by Koolhaas and L^eijer (see p. 26), Praak also gave the results obtained on another sample of derris prepar^^d in the Laboratory for Chemical He- search at Euitensorg, Java, and submitted to the laboratories of Seil, Putt, and Rusby in Nev/ York and of Salairion and Seaber in London. The spinpla, with approximately 6.5 percent of rotenone end 22 percent of total extract, w.as analyzed by the methods of Seil (see p. 25) using longer extraction time, Jones' and Graham (72_)i Seaber ( ill ) , and Koolhaas (78). The Laboratory for Cheviical Research and Soil, Putt, and Imsby, ~ when applying the method of Jones and Graham, obtained practically iden- tical results both for pure and crude rotenone. The Laboratory for Chemical Research found that the values for crude rotenone by the Jnnes and Granam method were almost the same as those for pure rotenone by the method of Koolhaas. The method of Seaber gave very different results when applied in the Dutch East Indies and in the commercial laboratory in London - 40 - The method of Sell gave results conridt=!ra'bly lower than by the other methods. However, another sar.plo prepared "by the Butch laboratory and analyzed "by Sell, Putt, and Rushy £:ave slightly higher results by the Sell method than "by the Jones and Grah-im method. Although Braak "believed the method of Koolhaas to ^ive figures closer to the actual rotenone content, he stated that it would "be most desira"ble for the method of Jones and Graham to "be applied universally, since it satisfied all reasona'ble demands for reproducibility in various laboratories and the chance of its general accept- ance seemed better than for any other method. Begue (5) in 1939 stated that the technique of Worsley was one of the best gravimetric procedures for rotenone determination, and he advised its use for derris root. Kumerous methods for evalijatin;- rotenone-bearing plants were criti- cally reviewed by Guillaujne and Ke'rve (53) in 1939. All crystallization methods were said to be impractical for determining small quantities of rotenone, as crystallization is not effected. Increasing the size of the sajuple was said to diminish th? sensitivity of the method. Such methods were said to be inapplicable to leaves end fruits. Colorimetric methods or a determination of inethoxyl groups v;as recommended. Georgi ^& Teik (5?) in 1939 stated that the findings of Seaber ( ill ) on decomposition of rotenone. after prolonged boiling with carbon tetra* chbrid^ had been confirmed in fneir laboratory. Consequently they had dis- continued the use of this solvent and substituted room-temperature extrac- tion wifi chloroform. Sxtraction and cr^'stallization were carried on as in the methods of Beach (3) anc^ Jones and Graham (72), except that the extract was allowed to crystalline for 2 days. One gram of pure rotenone was added to extracts of roots containing 6 percent or less of rotenone. After sepa- ration of the first crop of crude carbon tetrachloride solvate, the volume of the mother liquor was reduced to 15 cc. and placed in the refrigerator fpr 1 day to obtain a second crop of crystals. They were added to the first crop and the purity of the whole was determined by the alcohol- recovery method of Cahn and Bosun (13). Graham (49) in his 1939 report to the Association of Official Agri- cultural Chemists, described the results of collaborative analyses of five samples of derris, timbo, and barbasco by the crystallization method (72) and by the titration method (§7). Decolorizing carbon was used in the extraction flask as a result of the findings of Graham (46) on this point. Results were in fairly close agreement except on one sample of derris root of low rotenone content, which gave poor results by both methods. It was recommended that the cr>-stallization method, adopted as "tentative" in 1937, be amended to include the use of decolorizing carbon and be adopted as "official, first action." Graham (48) in 1940 reviewed the results cf the collaborative analyses just described. He pointed out that the addition of decolorizing carbon re- sulted in higher values for cube samples, and stressed the necessity of using the multiple-extraction procedure with chloroform for sajcples with a ratio of rotenone to total extract greater than 40 percent. - 41 - In 1940.1''e.i.jer arid I^oolhaas (69_) r'sscri'bed the method in use at the Laboratory for Chemical Research, Buitenzorg, Java, for determining rotenone in derris root. This is a modification of the original method of Koolhaas (78), and. since it is one of the more important methods now in use it is presented in some detail here: "Extraction. ?ifty gr.ams of powder (at least 75 percent must pass an 80 mesh sieve) are percolated with ether in a Soxhlet , without a thimble but with cotton wool at the bottom, for 65 hours. The heat used for boiling the ether comes from a SO-watt electric bulb. 'Tistillinj- off the Solvent. The ether is distilled off in a 100 cc. centrifuge tube on a water bath; the tuba is filled with the ether solution by means of a dropping funnel. Th9 rotenone which may have seps.rated during the extraction is transferred quantitatively to the tube, and the flask is rinsed, out with ether a few times. The final solution in the tube must be 25 cc. The tube is then tightly closed with a cork. "Crystallization of Rotenone. The tube with the ether extract is kept at room temperature for 1 day axxA then in a refrigerator for 2 days. The mother liquor is poured into a 50 cc. Hlrlenmeyer flask and the remaining crude rotenone is broken up with a spatula, with the addition of 10 to 15 cc . of ether. The tube and the ^rlenmeyer flask are closed with a cork and placed in the refrigerator for another day. "Determination of Rotenone. The rotenone is centrifuged for 3 to 5 minutes at 3500 revolutions per luinute and the supernatant liquor is added to the mother liquor in the 3rlenmeyer flask. Tine centrifuge tube with the crude rotenone is dried for 10 minutes in a water bath at 70° C., and after a slow current of air has been passed into the tube, it is dried in vacuo on a boiling water bath for 15 minutes. After cooling in a desiccator the tube is weighed. ♦ * * The purity of the crude rotenone is determined by the melting point, using an empirical table in which the correlation between the purity and melting point is given. If the melting point is lower than 140*^ C , the mass in, 'the centrifuge tube is treated with another 10 cc . of .ether, centrifuged, and dried, and the melting point is again determined * * *.■ The autnors determine tho optical rotation, from which the purity of rotenone can, also be determined. A correction is made for the amount of rotenone dissolved in the ether of " .the mother liq\i.or and the ether used for washing. For each cubic centimeter of ether used 4.2 mg. of rotenone are added to the amount of pure rotenone. If loten- ona has separated from thei^other liquor to which the wash ether has been added, after standing in the refrigerator for another night, it is centrifuged off and added to the crude rotenone * * ». ■ , . ■ ; ' ■ . ' . ( ■» . -, - 42 - These author.^ attempted .to determine "by two schemes the amount of rotenone left in the resin after crystallization. The first involved extraction of the nonrotenone resins with petroleum ether and cyclohexane and crystallization from carbon tetrachloride o: the rotenone in the residue, while the second method was "based on adsorption on activated fuller's earth and washing cut with benzene, the evapo:'ated residue being crystallized from carbon tetrachloride. From a mLnber of samples of resin a ran^e of from 4.5 to 17.0 percent (with two exceptions), with an average of about 10 percent, of the amount of the original rotenone was recovered. Results of the anal;' ses of 40 samples of derris root by the authors' method and by that of Jones and G-rah?m (72^ were compared. T"ne results for piiTe rotenone by the authors' method were in close agreement with those for cruJe rotenone oy the latter method. It was found that finer zrinding in gen?ja.l resulted in higher values for both rotenone end total ether extract. Moi.ier and Koolhaas belie/ed that samples with a ratio of rotenone to total extract higher than 40 percent, which v;ere said to be common in the Dutch alast Indies, cculd be handled satisfactorily either by finer grindin^^ (75 percent through a 200-nesh sieve) or by extraction several times with chloroform. They stated that a satisfactory uniform method for rotenone might be based on the Jones and Graham method, provided the values for crude rotenon? were used and this method was adapted to samples with a high ratio of rotenone to total extract. The authors compared results for purity of the carbon tetrachloride solvate by polarizat?, on, alcohol recovery, and the titration method of Jones (S7). The results were generally in good agreement; alcohol recovery in general gave the lowest values, polarization highest, and titration between these two. The degree of purity when deter- mined by the melting point was said to be generally higher than when deter- mined either by polarization or alcohol recovery. As they experienced difficulty with the titration method when 20 percent or more impiirity was present and as the polarization method was even less time-consuming, the authors preferred the latter method. The figures previously given by i'eijer (87) to show fne effect on the rotenone content of drying the sample were repeated. Keating the sample above 50° C. before analysis was strongly discouraged. In further work on the evaluation of rotenone-containing plants, I'artin (83) in 1940 described the procedure for determining rotenone in derris, which involved some modification of previous methods. Sufficient root to give about 5 gm. of resin was extracted by hot percolation with ethyl acetate for 3 hours in the apparatus already described under methods for Total Extract. The resin, freed of solvent, was dissolved in 100 ml. of a mixture of benzene and ether (25 percent by volume of benzene) and extracted rapidly with 50 ml. of 2-percent potassium hydroxide and then with two lots of 5-percent potassium 'tiydroxide. Water was then immediately added to the benzene-ether solution to dilute any residual alkali. The combined alkaline extract was washed with benzene-ether and the wash added to the main solution. The combined benzene-ether solution was then washed three times witn water and dried with anhydrous sodium sulfate, and the solvent was removed. The resin was dissolved in carbon tetrachloride and the rotenone solvate crystallized in the usual way. Determination of tne - 43 - purity of the complex was ca^^ried out by a polarimetric method. The optical rotation of a 4-percfcnt roluticn cf the solvate in "ben'rene was determined, and the percentage of rot3non^ tnen calculated from the rotation of pure carbon tetrachloride solvate in ben'^ene. It v,as stated that impurities present were likely to rhov/ a specific rotation ap-pro'cimately one-fourth that of rotenons. x]-.3 error involved v/as said to be small but mi,5;ht be allov/ed for if it was ccnsicieiGd necessary. Valiies obtained cy this method, in general, agreed with t'lops cbtai.ied by the dichloroc-icatic acid titration method (67). The Imperisl Ins^ibnte (57), in a surv.^y of insecticides from plant materials in 1940, oriefly discussed the evc.luatio-i of derrls and cube. It was stated that results for rctenone depend.ed on the solvent, the number of extractions, tha tempe'^ature and leijf:th of time of extraction, as v/ell as on the finenerss of gri-iding and moist'ore content of the sEjnple . The need of a standardised method v/as stressed. In the Official and Tentative I.:Hhcds of Analysis. of th3 Association of Official AfiTiculo\iral Chemists (Z) ^re f cllov/in^'; method for determining rotenone, that recornisnded ^y Graham (ji?) , i^ girexi as "official, first action": "weigh 30 g. (if samplo contc.ins inors than 7,^ rctenone use a quejitity that v/ill give 1.0 - 1.5 p. of rctenone in the 200 ml. aliquot) of finely powdered rco-b aiid 10 g. of decolor- izing carbon into 500 -nl. glass-stoppersd lirlerjneyer flask. Add 300 r.l. of cnlor->form neesurod at definit-=! roca temperature; place' flask on shakir.g machine and fasten stopper secure].?/. Agitate vi;3orously for not less th^n fo-jr ho-ars, preferably interrupting shaking v;ith overnight re«t (or flask iosy be shaken continuoi''sly overni,5^t). P-Sinove flask irom machine end alJ.ov; to cool in refrigerator for at least an hour. Filter mixture rapidly into suitable flask, usiiifj fluted paper without suction and keeping funnel covered with watch glass to avoid loss from evaporation, titoppor flask and adjust temperature cf filtrate to that of original chloroform. "Transfer exact?.y 200 ml. o^? this solution to 500 n.l. Pyrex 3rlenmeyer flask and distil until only about 25 ml. regains in flask. Transfer extract to 125 ml. 3rlenme:'-er flask, ■'.ising carbon tetrachloride to rinse out the 500 ml. flask. Svaporate almost to dr7,rress on stea'a bath in current cf air. Then remove remainder of solvent uiider reduced pressure, heating cautiously on ste-Mf; bath when necessary to hasten evaporation (suction may be applied directly to flask). Dissolve extrs-ct in 15 ml. of hot carbon tetrachloride and again, in a similar manner, remove all solvent. Repeat with .another 10 - 15 ml. portion of hot carbon tetrachloride. (This treatment removes all chloroform from the resins. The chloroform extract is visually completely soluble in carbon tetrachloride. If small quantities of insoluble material are present, the purification procedure doscribod later will eliminate them. However, if large qua.ntity of insoluble residue should remain when -extract is dissolved in first portion of carbon tetrachloride, it should be filtered off and thoroioghly - 44 - washed with further portions of hot solvent, aftsr v/hich the filtered solution plus washings should oe treated as directed above for removal of chloroform.) "Add exactly 25 rl . of carhon tetrachloride and heat gently to completely dissolve extract. Cool flask in ice bath for several minutes and seed with a. fev; crystals of rotenone-carbon tetra- chloride solvate if necessary. Stop-oer flask and sv/irl until crystal- lization is apparent. If at this stage only a small q^iantity of • crystalline material separates, add an accurately weighed quantity of pure rotenone estimsted to be sufficient to assui'e that final result, expressed as pure rotenone, is at least 1 g. Then warm to effect complete solution, and again induce crystallisation. At same time prepare a saturated solution of rotenone in carbon tetra- chloi^dB for washing. Place flasks containing extract and v/ashins solution in ice bath capa.ble of maintaining temperature of 0° G. and allow to remain overnight. "After 17-18 hours in ice bath, rapidly filter extract through weighed Gooch crucible fitted with disc of filter paper, reproving flask from ice bath only long enough to poiu" each frac- tion of extract into crucible. Hinse residue of crystalline material fvoir, flask and wash under suction vrith sufficient of ice- cold saturated solution (usually 10 - 12 ml.) to remove excess mother liquor. Allow crucible to remain under suction for about 5 minutes and then dry to constant weight at 40° C. (about an hour). The weight obtained is 'crude rotenone-carbon tetrachloride solvate'. "Break up contents of crucible with spatula, mix thoroughly, and weigh 1 g, into 50 ml. -^rlenmeyer flask. Add 10 ml. of alcohol that has previously been saturated with rotenone at room tempera- ture, swirl flask a few ninutes, stopper tightly, ajid set aside at least four hours, prefer-^bly overnight, at the same temperature. J'ilter on v/eighed C-ooch crucible fitted v/ith disc of filter paper. P-inse crystals from flask and wash under suction with solution of ethyl alcohol saturated with rotenone at tempera- ture of recrystalli^ation lea. 10 mi will usually be required). Allow crucible to rer:vain ur-der suction 3 to 5 ninutes and then dry at 105° G. to constant weight, which should be effected in 1 hour. ""'ultiply weight expressed in gra:nnn b^- v;5ight of the 'crude rotenone-carbon tetrachloride solvate' and to prodi\ct add 0.07 g. which represent b correction for rotenone held in solution in the 25 ml. of carbon tetrachloride used in crystallization. If any pure rotenone has been add.ed, subtr.-^ct its weight from vilue obtained. This gives the weight of pure rotenone contained in bhe aliquot of the extract, representing 20 grams of the sample. "Alternate extraction procedure. - If sample is one in which ratio of rotenone to total extract is greater than 40/j, use quantity sufficient to contain 1.0 - 1.5 g. of rotenone and successively extract four times with CHGlr^, using 200 ml. each for second to fourth extractions, filter after each extraction and return marc - 45 - to flask for extraction with fresh solvent, i'^inally combine extracts, evaporate almost to riryness, anct proceed as directed alDove, be^;:innin£; at point where aliquot has "been evaporated almost to dryness." The same commercial sources that supplied information on the deter- mination of total extract also discussed the methods used "by them for the determination of rotenone (private comm\ini cat ions ) . Two of them, IlcCormick and Company and John Powell and Company, employed the official A. 0. A. C. method. The laboratory of S. B. Penick and Company, regularly used the official m?^bod but also occasionally employed a modif " - ation for compara- tive purpos'^'Cs. Thn s method, which depended on measuri^.£: the carbon tetra- chloride of crystallisation and assumed that rotenone is the only solvated material present, was as follows: Place ths crucible containing the crude solvate (dried to constnr;t weiffht) in a lOO-cc. beaker and weigh. Add 5 cc. of alcohol (or acetone) to the material in the -nrur-".ble and keep in a modcrav.ely warm place, where evaporatr'.on /'■j.ll prcT-sed iJowly, until all the solvent has evaporated. Repeat this step ,' /;ice and then dry the beaker and contents at 100*^ C. 'co con!;i;r.nt weight. This procedure removes the carbon tetrachloride of crystallization, which was combined in equimolecular ratio with the rotenone in the solvate. The difference in weight divided by 0.281 and multiplied by 0.719 gives the weight of rotenone. . Another firm, Derris, Incorporated, preferred to use acetone as the extraction solvent, claiming that this solvent extracted the resins and rotenone in less time than any other solvent tried. The method was briefly as follows: ?rom 40 to 50 gm. of the coarsely vround or 25 to 50 gm. of finely powdered root is extracted in a Soxhlet with acetone. (If powdered root is -jsed, it is mixed with an equal voliune of fine sand or sodiiyn chloride.) At the end of TA hours the extraction flask is replaced by another containing fresh acetone and the extrac- tion continued for about 20 minutes, or until a negative color test (I>arham) shows that the marc is depleted of rotenone. The combined acetone extracts are then concentrated to about 10 cc, when two or three a.'iditions and evaporations of 25 cc. each of carbon tetra- chloride are made to remove the acetone. One hundred cubic centir^-eters of carbon tetrachloride and about 3 gm. of Celite are added to the residue and the whole is heated to boiling and filtered by suction. The residue is washed with two or three lots of about 25 cc. each of hot carbon tetrachloride. The carbon tetrachloride solution is concentrated to about 25 cc. and transferred to a 50-cc. graduated cylinder with ground-glass stopper. The volume is adjusted to 40 cc. with carbon tetrachloride and the cylinder placed in the refrigerator to remain overnight. The filtration, washing, and determination of purity are carried out approximately as in the official A. 0, A. C. method. - 46 - HesTilts by this method were said to "be in agreement with those "by the various published proced-ores, including the official' A." 0. A; C, method. L'isouBsioo ' It is generally agreed that derris and cube root should be finely grovmd and air-dried before extraction. Several investigators (71, 87^) have shown that drying at elevated temperature is detrimental to the determination of rotenone. ' ~ - One of the best solvents for extracting rotenone for analytical .purposes is chloroform. Extraction at room temperature seems to be pre- ferred by most workers. This method is rapid, yet it extracts less extra- neous material and involves less decomposition than the use of other sol- vents or higher temperatures. For most roots the aliquot procedure appears to be entirely suitable, but for roots with a high proportion of rotenone, as pointed out by Jones and Graham (71) and I^IeiJer and Koolhaas (89_), a more exhaustive extraction must be resorted to. For this reason the alter- native multiple-extraction proced.ure is included in the official A, 0. A. C. method (2).^' Crystallization from c.arbon tetrachloride as carried out by most of the present methoc-s may be consi-iiered to be reasonably complete. It is possible, as indicated by the work of Cahn, Phipers, and Boam (17.), and of l.'ei.jer and Koolhaas (89) , that in some few samples, particularly those with a very low ratio of rotenone to total extract, a small proportion of the rotenone r^^.T.ains unaccounted for in the mother liquor. 'rt"hile the reviewer (66) believes this to be primarily a result of greatly retarded rate of crystallization in certain types of extracts, other investigators (17) state that it is due to an actual solvent effect of other constituents. Whichever may be the case, anything that can be done to increase the relative proportion of rotenone v/ill aid in producing more nearly complete crystallization in the time allowed. The addition of pure rotenone (13, 65), the treatment with decolorizing carbon ( 130 , 46) , and the removal of alkali-soluble material (64) all tend to accomplish this result. In the reviewer' s opinion, when these schemes or combinations of them are employed, samples in whicn rotenone remains unaccounted for in the resin will be encountered, only rarely, and even then results will not be greatly in error. If derris samples with a very low proportion of rotenone to total extract are to be encountered regula'rly on the market, alkali extraction may become a necessary step. Such roots as these usually contain a large proportion of toxicarol and other phenols, and their removal undoubtedly improves the crystallization of the rotenone. 8 Recently a sample of derris root encountered in the laboratories of this Bureau was not completely extracted even by this method and it was neces- sary to resort to hot extraction. This sample contained about 10 percent of rotenone and 24 percent of total extract. 47 Kost investigators nov; feel th£«.t pure rather than crude rotenone should be reported, vvith regard to the oes'o method of determining purity there is sorje dif.'^erence of opinion. The alcohol-recovery method U_3, 63) ir, the most" u^-^ea , although it generally gives slightly lov/er \-alues than othnr methods. The titration scheine (67) ir^ay gi"V2 more accurate vJ-uos, but difficulty has teen experienced in apo lying it to low-rotonone roots. Polar- ization ( ill , 89, ISO }' is used out p-^obaoly gives values that are slightly too high. According to Meijer and Koolhaas (69) the purity determined hy melting point is generally higher than "by polarization. In any of th-3?e methods more nearly the correct value will "be obtainaa the purer the original solvate. Since the three methods already discuf^sed for treating the extract before crystal- lization .— namely , add'^.tion 01 rotenone, use of carbon, and alicali extraction — in general' res-.ilt iri a purer solvate, they are- to be recomiEpended f i oia this sta-'-cpoint as well as from that of improving the completeness of crystallization. Thus it appears from work reported by the Imperial inctitute ( 53 ) that alkali extraction might obviate much of the difficulty enccur.tered by some v.rorkers in tne ut^e of the titistion irethod with low-rotencne roots. Without doubt the procedure for the deter.Tiinatr'.on of rotenone that involves room-temper.atu2 e extraction with chloroform and crystallization from carbon tetrachloride, as exeiiiplified by the official A. 0. A. C. method (2), is nov the most v/idely used. 4^ D3GU2Liri AITD HDT-NOlIE PLUS DZGUSLIN The Durham Test A test vfidely used in work on rot-.none-containing plants is that based on the color recction discov.ircd "by Darham in 1902, in which treatment of certai constituents of derris root \vith nitric acid followed "by aminonia v;?.s foujid to produce an evanescent "blue-^reen color. Ishikawa (50) in 1916 independently discovor^id this reaction, using sodiur. hydroxide as the "base. According to Tattersfit^^ld and Hoach ( 12^) and Giralettc (3B) , who descrited Durham's work, concentratsd nitric acid v/a? added to solid rotenone, or rosin, and this mixture was treated directly v.lth strong ajanonium hydroxide. In this form the test was qualitative only and was unsuitatle for delicate testing oecause of the violence of the nt^utralization. Payer a:id Hincrbein (92) in 1931 described a qualitative test similar to that of DLirhpjD. To 2 cc. of dilute acetic acid extract of the sar.ple, 2 drops of fuming nit ric acid were added; the mixture WaS diluted with 10 cc. of water and then made alkaline with sodiu;n hj^'droxide. The color produced passed quick- ly from green to brown. The green color produced with aaaonia was said to be more stable than that obtained with scditLm hydroxide. In 1933 Jon( s and Smith (75) modified the I>urham test in an attempt to render the blue color more pemanant and make the test more nearly quantitative. They used the following procedure: T& 1 CG. of an acetone solution of rotenone (or a plant extract) 1 cc. of 1 -♦. 1 nitric acid is added, and the mixture i? allowed to stand for l/2 minute. It is then diluted with 8 to 9 cc of water and 1 cc. of strong amaoni\im hydroxide is added. A blue color is produced wMch is ainost identical with that given by bromothymol blue indicator at a pH of 7.2. It was said that as little as 0.1 mg. of rotenone could be detected by this method. Ey preparing a series of solutions contairing different concentrations of bromothymol blue in a buffer of pH 7.2 and standardizing these against the colors produced by different amounts of rotenone, it v/as possible to use the test to make a rough estimate of the amo-'ont of rotenone present. Since approx- imately the same intensity of color was given by deguelin, whereas toxicarol gave alm.ost no color, it wgs believed that the test gave a rough value for the total rotenone plus deguelin content of plant extractives. Sodium and potassiion hydroxides, or sodi\:m and potassium carbonates, used in place of the ammonia, were also foiond to produce the blue color. Vhen the nitric acid was partially neutralized with sodiixm bicarbonate a:id the neutralization was completed with a stronger base. Thitt'dolor w^is sli:;^htly more permanent. The first reported semiquantitative application of this test was made by Jones, Campbell, and Sullivan (70) in 1935, in working with a series of samples of several species of xeph rooia. i.ctracts that gave the test were rated in three grades aependirg on the depth of color produced. Fischer and Hitcohe (2^) in 1935 made a study of the tests proposed by Peyer and Huncrbein (92) and by Jones and Smith (75) in a further attemot to render the reaction more nearly quantitative. The test, carried out in 49 essentially the same. manner as already describt-d except that the diluted re- action mixture vcs' cooled .to 0*^ C, "before addition of the annoriia, w^s as • follovs: • To, prepare color standards, 50 ng. of malachite green is dissolved in 1 liter of water, a portion of the solution is. diluted' to douhle the volune and the procedure is repeated un- til six solutions of successively greater dilution have "been prepared. In general, in the analysis of rotenone and rotenone- rich extracts a 0.4-percent solution in acetone is, needed. One part is diluted to double the volume and another to four times the volume, so that the three solutions contain 4, 2, and 1 mg. per cubic centimeter of rotenone or extract. At th© time of analysis 1 cc. of acetone solution is mixed with 2,cc. of dilute nitric acid (sp. gr. 1.2), allowed to stand 2 minutes, diluted with 7 cc. of ice-cold water, and. cooled to 0° , Two cubic centimeters of 2S-percent ammonia is then added rapidly, and the liquid is quickly mixed and compared visually with the standards. 'Estimations must be made not later than 3 to 4 seconds after addition of the ammonia. Orange-yellow color filters may be used in making the comparison. Jor a repetition of the measurements another series of concentrations of the unknown sample should be chosen. Values obtained may be plotted graphically with the standards if desired. As the end value of an analysis the middle value from three observations at different concentrations may be used. It should not differ more than about 10 percent from the end value of a second analysis. In some cases extraction vlth benzene followed by evaporation and solution in acetone removed impurities that interfered with the measurement of the color. In the presence of many substances the appearance of the yellov; color with nitric acid was v-ry'much retarded, and in these cases it was necessary to add a drop of f-uming nitric acid to initiate the first reaction. The colorimetric method of Jones and Smith (75) was regarded by Groudswaard and" Timmers (^ in 1937 as unsuitatle for the estimation of rotenone, because the intensity of the color produced depended on the temper- ature and because various tints interfered wl':h the estimation of the color. Pozz.i-Sscot -(98) in 1937 stated tliat bases other than ammonia produced the final blue color in the Durham reaction. Even organic bases such as triethanolamine produced a blue-green color. Sievers and coworkers (ij^ in 1938 extended the semiquantitative . use of the Jones' and Smith (7^ variation of the Durham reaction to several hundred samples of Tephrosia virginiana . Extracts v/ere rated in five dif-. ferent grades depending on the degree of color, and two investigators work- ing independently gave substantially the same rating to the majority of samples. Th9 Durham reaction has been adapted to the qualitative testing of mineral-oil fly sprays for rotenone and deguelin (l25). Directions for the test were as follows: 50 "Trsmsfer 5 cc. of the material to a large test tube, add 2 cc. of concentrated ritric acid and shake for 30 to 30 seconds, . dilute immediately with 20 cc. of v.'ater. Close the tube and shake to disperse the oil in the aqueous solution; allow the oil to -separate, which should not require more than 30 to 40 seconds, and add 1 or 2 cc- of amnonf.a poured dov.Ti the side of- the tube. The presence of rotenone or derris extract is indicated by the formation of a fugitive blue color." Quantities of rotenone as small as 2.5 mg. in 5 cc. of a mineral oil-pyre thrum extract were said to have been detected oy this method. Guillaume and Herve {^) in 1939 described a modification of the Durham test which rendered the color slightly more peruanent r.nd permitted a ro-ughly quantitative estim?tion. Tnis schame involved r.dding only a few drops of nitric acid to the acetone solution of rot»='none or exoract, covering with a layer of toluene and allowing to str.nd in vacuo at -8° tc-J.O<^ for 1 ■. hour. The blue color then obtained by addin-^ a fe'-f drops of £-?jaonia persisted unchanged for about 1 minute. Coloro produced s-r-multaneously from the s-ample and from stand- ard soliitions of rotenone v;ere compared rrpicly in tert tub^s.' In this form the method was said to be sufficiently precise end sensitive for many purposes. The G-ross and Smith Test A more useful color rea-:;tion than that of Durhr.m from the quantitative standpoint was discovered in 1934 by G-ross and Smith (50), It involved treat- ment of an acetone extract of the sa.nple with alcoholic alkali followed by nitric acid containing nitrite. A rather permanent red color was given by both rotenone and degaelin, and also by din^dro rotenone. Briefly the proce*- (iire was as follows; To 2 cc. of an acetone solution or extract containing 0.05 to 0.30 mg, of rotenone rjer cubic centimeter, add 2 cc. of 10* percent alcoholic potassi-um hydx-oxi-de solution, and allov; to stand at 20^ C. for 2 minutes. Then add 6 cc. of a nitric acid- sodium nitrite mixture containing 1 volume of concentrated nitric acid to 1 vol-ume of aqueous sodium nitrite having 0.25 gm. of sodium' nitrite por liter. Mix, cool to 20° C, and allow to stand at this temperature for 15 minutes. Comp,?re visually with standi ards containing pure rotenone prepared at the srjne time. When applied to samples containing only rotenone, the results agreed with those by the gravimetric methods. Applied to derris a:id cube samples, the results were 50 to 100 percent higher than for rotenone alone, owing to the presence of deguolin. The method was applied to rotenone spray residues on fruits and foliage. In using the Gross and Smith test for total rotenone and deguelin in several sajruples of derris root, Tattersfield and Martin (igO) employed two methods of extraction. In one method the saraples were extracted with acetone and aliquots of this extract were diluted to the proper concentration for the 51 test. In the other procedure the samples were extracted vdth ether and the dried extract vias dissolved in acetone and treated as in the first method. Colors developed from extracts by the second method were more readily matched than those obtained from the first method. Ambrose and Eaag (j[) in 1936 shortened this test for use in detecting rotenone and deguelin in the excreta of animals fed rotenone and derris. The material was extracted with ether and the evaporated extract taken up in acetone. To this solution was added one-third its volume of freshly prepared 10-percent alcoholic potassi\ain hydroxide, and the resulting mixture warmed. A vane-red color developed to a maximum in about 15 minutes. The test was said to be sensitive to 1 part of rotenone in 35,000. Goodhue (4g) in 1936 described a more serviceable modification of the Gross and Smith test, which has Jbeen widely used in subsequent work. Sulfuric acid was substituted for nitric, the concentration of the alcoholic potassium hydroxide was reduced, and the nitrite necessary to produce the color v/as added in the alkali instead of in the acid. The method v;as as follows: Prepare the following reagents: (a) Kix 1 voliime of sulfuric acid (sp. gr. 1.84, free from nitrous acid) with 3 volumes of water, (b) Dissolve 1 gram of sodium nitrite in 10 cc. of water and dilute to 1 liter with 95-percent alcohol, (c) Dissolve 40 grams of potassium hydroxide in 100 cc. of water, (d) Mix 1 folnme of -reagent (c) and 7 volumes of reagent (b) . Prepare this solution fresh daily. Proceed as follows; Prepare an acetone extract of the sample containing from 0.005 to 0.25 mg. of rotenone per cubic centimeter and pipette 2 cc. into a dry test tube. Add 2 cc of reagent (d) and place the tube in a water bath at about 25° C. for 5 minutes. Add 5 cc. of reagent (a), stojoer, shake, and place the tube back in the water bath. The color reaches a maximum after aoout 15 minutes, and then remains unchanged for 2 hours. Determine the amount of rotenone by comparing the color with standaiids prepared at the same time from known quantities of rotenone. The turbidity or brown color which sometimes developed during the analysis of crude plant extracts was removed upon extraction of the final mixture v/ith a small portion of ether. The red color due to rotenone and deguelin was not removed. In this work comparisons were made v/ith Lovibond color slides in a roulette comparator. The small amount of blue which accompanied the red was filtered out by a dichromate filter for easier matching. By this modified procedure the sensitivity of the original test was increased 20 times. The specificity for rotenone and deguelin remained the same. Deguelin was said to give the same amount of color as did rotenone. This method was criticized by Gouds'-vaard and Timmers (42) in 1937 on the same grounds as the Jones and Smith (75) test-namely, dependence of color in- tensity on the temperature and interference of various tints with the estimation ' 52 of the color. On the other hp.nd, the aethod was said "by Schonberg (iiO) to present several advantages over the crystallization method. Among these were the fact that it would determine very small quantities and that it was rapid and practical. Furthermore, the oxidation of rotenone to dehydrorotenone could be followed by this colorimetric method but not by crystallization methods. Cahij, Phipers, and Boam (jLZ) in 1938, in an extensitre study of the com- position of derris root, used this colorimetric method, making color comparisons in a Duboscq colorineter. They proposed to call the result obtained the "G-ood- hue value"; this was the percentage of rotenone that the material would contain if roti^none virr? the only ingredient siving a color in the test. They found these values to bear a definite relation to the rotenone content of derris extract. For ordinary extracts the relation was as follows: Goodhue value = percent rotenone -^ 22-3, This relation did not hold for Sumatra-type extracts, vhich usually had C-oodhue values of 10 to 15. As mentioned in the section on Rotenone, this relation was used by Cahn^Phipers , and Boam to determine approxi- mate rotenone contents by the color method. According to these wri,ters,,8x>t.enone and deguelln ^ . .were the only substances (kmown at that time) in derris extract vrhich gave this color test. They found that the color given by deguelin was only four-fifths the intensity of that given by rotenone. Consequently, they stated •tiiat the "excess" . value a^ove the actual rotenone content mast be multi- plied by .1.S5 to indicate the degij.elin content of an extract. Using this value, they concluded the deguelin content of derris extracts to be 27 ^ 4 percent, except Sumatra- type extracts, which -contained 9 to 15 uercent of degaelin. Again in 1939 Cahn and Soam {^S) discussed the determination of approxi- mate rotenone content by means of the Goodhue value. This scheme has been m.entioned in the section on Rotenone. In a study of colorimetric procedures in 1939. J ones (68) used this method and made color comp^^ri sons in a neutr&l wedge photometer using a filter with its optical center at 0.56 micron. This neans of measuring the color had been in use for Bome time by Goodhue and '-oy Cassil in the laboratories of this Bureau. Begue (_5) in 1939 recommended this method for the analysis of products containing; less than 1 percent of active principles. Guilla-ume and Herve ( 53) in 1939 described and discussed Goodhue's modification of this method. They suggested that for this test a 1-gm. sample be extracted with ICO cc. of acetone in a Soxhlet or a Kumagava"(8l) apparatus for 5 to 8 hours. They also found that complete exhaustion of 1 sjn. of powder was obtained oy agitating with 100 cc of acetone in a "flask .for 2 hours. They found it unnecessary to prepare a standard solution of rotenone each time the test v/as made. Instead, they made a 0.05-percent solution in acetone and kept it in the dark in ice (or at laboratory temperature) for use in a large number of deter:riir.ations. The procedure used for the development of the color was essentiall,," t'le same as that already described. Although this method gave results on freshly prepared powders agreeing with those of the blue color test and the methoxyl method, it gave lower results on old powders than the other two methods. 53 (Joodhue and Haller (^2) in 1940 used this method for the estimation of dihydrorctenone in the liydrc? -'nation products of rotenane. Altnoufljh this derivative of rotenonr hrs rol: "been foand in dtrri3 £u~id cu-je roots, the modified procedure is equally rpplicahlG to the det^niAnatio-i of rotenone and dc^jielin in extracts of these plants. The procedure was practically identical vdth Goodhue's earlier modification (4C0 of the test eycept th;:it 6 cc.( instead of 5 ccO of dilute sulfuric acikl was used follov/ln/? the addition of ali-iali and the solution vas then maintained in a cold-water bath at 15 to ?-00 C. for ahout 5 minutes (instead of at 25^ for a lon^^jer period). Howe-'rer, the rneasurement of the color intensity was ir.-proved. Thi3 pc:.?tion of the procedure was as followst "The use of a photometer has "been found to "be thf^ most tconir-tte method of con:paring the standards and the unknov.TiS. Tha colors crj:i be developed dix-ecoly in selected test tribes v/hich fit the photometer, or they can be dcveloxicd in sny test tube and poured into a rpecial '.-cell. A Brice photometer which g-ives percentage transniiscion as ai.rect readings has been found satiefactory. Glass filters (CorrJ.ng, Z,5 mm., No. 430, dark shads blv.e-groer) were used. The blanic is taJcen as 100 percent trajismission, rnd the photometer is therefore adjusted to give a, reading of 100 vdth the cell containing the bian'x: in place. The readings for the standards and -unknowns --rhiah are next obtained are therefore in percent trensml3F.ion. "*»**'the color for rotenone has b^en four-.d by Cpssil (unpublished) to follow Peer's lav*-''-^/ A plot is therefore made on 3emilf>-^a^ithmic paper with transmission as orainato (logaritimaic) and concentration as -sbaclssa (arithmetic), and a straight line is dra-.vn from the point of zero concentration and 100 percent transmission to the point determined by the concentration and the transjuission of th'^ stai:dard. The trazis- mission of the unknown having been determined, its concentration can be read from the curve and the percentage calcuirted." Cassil (19) in 1941 used tne red color test for the determination of derris-du.st rec.idues on cabbages. The residues were extracted by washing the leaves with chloroform, the extract evaoorated, and the residiue dissolved in hot acetone. The acetone solution was chilled to remove wft.T.y material, and the filtered solution was used for the color test. The colors v^ere ccmorred with those from extracts of kno^'m quantities of the derri^' root actually used in the original dusts. Comparisons of color intensity were made in a photoelectric photometer. 54 Gravimetric Methods T^ucei, MiyaJiTia, ard Ono ( 118) in 1933 developed a gravimetric method fOr detemining rotenone anddeguelin. This nethod, which has been "briefly discussed under Hctenone, is "based on the fact that rotenone and deg^aelin are readily oxidi:2ed under alkaline conditions to rotenolones and degaelinols, respectively, vhich are quantitatively dehydrated by alcoholic sulfuric acid to the hi,^hly in- soluble dehydrorot-enone and dohydrode^uelin.- r.ie mixture of d^hydro coiapounds gives a value for total rotenone plus deg-aelin. A value for dc^elin alone Was obtained by subjecting the dehydro co^rjounds to aporopriate cat-Jytic hydro genat ion, vhich con-'-erted the dehydro rotenone to the alkali-soluble isodinydrodehydro rotenone (dehydrorotenonic acid) while the dehydrodeg-ielin reraintd unchanged. In the original r.ethod much of the rotenone was first cr:,"stallized out. Die nethod is briefly as follo^^'s: The rotenone is first cr-'stellized fron an ether ertract in the uoual v.^ay. live grar,s of th?- evaporated mother liquor is dissolved in 150- cc. of alcohol and 3 gm. of S-^Dercent alcoholic sodium hydroxide is adaed. 0:cygen is passed in at the rate of 150 cc. per minute for l/S hour. If air is used instead of ox^rgen, it is run through for 1 1,/?, to 2 hours. The reaction product is acidified vith 15 grn. of 50 — ercent alcoholic sulfuric acid, and 130 cc. of alcohol is distilled off on the ^^rater bath during 1/2 hour. The residue is refluxed another hour. It is nixed with 500 cc. of water and shak-r-n vj _^orour!ly v/ith SCO cc. of ether in a separetory funnel. The dehydro ceriv -tives are suspended in the ether layer, other materials dissolve in either thr water or the ethor layer. The entire mixture is filtered by suction, and the crystals are washed with 10 cc. of methyl alcohol. The yellow* needle crystals a.r9 dried at 100° and weighed. ' This value gives the weight of any rotenone not separated in the original ether crystallization plus the deguelin. A small quantity of crystals may also be obtained from the filtrate. Five-tenths ^.rsia of the mixture of dehydro compo-ands and 0,5 gn. of palladium-bariuT. sulfate catalyst are placed in 100 cc . of alcohol and mixed witri 3 c6, of 3-percent sodium hydroxide. The mixture is shaken in an atmosphere of hydrogen for 2 hours, and then filtered. The residue consists of dehydrodeguelin and catalyst, it is extracted vdth acetone, ajid evaporation of the extract gives the dehydrodeg'j^elin. Most of tne alcohol is distilled from the filtrate, vhich is then acidified with sulfuric acid and extracted with ether. Zvapor.ition of the ether extract gives the isodihydrodehydrorotenone. Danc':vortt, Buido, and Ba-'angarten {2Ji) in 1934 stated that the foregoing method might be theoretically unobjectionable but it was very trouolesor.e and time-consuming and required a la.Tr;e amount of aoparatus. Fischer and "itsche (?8) strted that the method was too bothf^rscme and time-consuming in its complete execution. They found it useful, howev r, for determining only the rum of rotenone and deguelin. Essentially the directions of Taicai ft fT> were used, but in v;ashing the sepi^.rated dehydro compounds the methyl alcohol w-^sh was follow<:.d with a small amount of ether. IVequently a 55 second or third methyl alcohol or eth.?r T±nr.e was necessary vhen the filtrate from the first was dark colored. In the case of plant a??terial9 inclined to resinify on oxidation, the sutstance was extracted \d.th ben?.ene and the evaporated benzene extract used for tne oxidation. The disturbing inaterialo were insoluble in benzene. In 19S5 Tatterjsficld and Martin (l?0) foimd that pure roter.one, vhen put through the first portion of the Tekei process, gave a yield of only about 80 percent of dehylrorotenone. The renair.der could be recovered from the solvents used for the final separation of dehydro compounds. Consequent- ly they modified this separation. After dehydration with alcoholic sulfuric acidf- the residue wos cooled for some time in ice, the crystals filtered by suction and washed successively with a little ice-cold ether, 100 cc. of distilled vater, end a few cc. of ice-cold mothyl alcohol. The crys-^.?ils wer^ dried at 100° C. and weighed. Tnc filtrate was then separated by add- ing 400 cc. of distillod water ard 200 cc. of ether. If a further yield of crystals was cbtain'=d, th<>y wer^) filtered, washed with a little ice-ccld methyl alcohol, and the weight added to that of the first lot. Tat t^i^rs field arid Martin in some cases used the whole ethir extr'-^ct of derris without preliminary separation of the rotenone, end in other casta the mother liquor from the separation of the carbon tetrachloride co-plex, v?hich they dissolved in the alcoholic mother liquor frora the purification of the retenone solvate. HightT results were obtained in general by the nicdifii^d method, either with or without removal of the rotenone, than by the original '2r'y:ei method made on the whole ether extract. In the folloving year the same investigators (f4) attempt 3d the quantitative preparation of "de-jaelin concentrates" of several samples of derris root, following the qualitative scheme used in the earlier i./ork of Haller and LalTorge (55). Twenty grams of the roots v/as extracted with petroleum ether, with rapid refli?.xin£;, for 55 hours. The evaporated extracts were dissolved in ether, and the rotenone was allowed to crystallif.e for 2 days in the ice chest and then separated. The ether filtrates were then extracted vath dilute potash, washed with water, dried cv ;r sodium sulfate, concentrated to a s:?:all volume, and placed in the ice chest for 5 days. Any further rotenone was separated. Tlie mother liquor fror. this separation was termed the "degij.elin concentr.^te'l" It probably contained most of the deguelin but may have contained other materials also. The values were somewhat higher than those obtained for dehydro compou:ids by the modified Takei method (l20) on the alkalii.n soluble portion? of the orif;iaal ether extracts. In t^iis work al§o the term "rotenont plus deguelin" was applied to a value calculated from the methoxyl content of the alkali-insoluble portion of the extract and based on the methoxyl content of rotenone and deguelin of 15.74 percent. In using Takci's dehydro meohod 'i^orsley (l3_3) in 1937 found the amount of wash solvents rccommcndv^d by Tatt^jrsfi.ad and Martin (l20) to be in- adequeto. Accordingly he ador;ted th.- folloving procodur^^i The cooled residue from the dehydration process was filtered farough a Gooch crucible and Washed with 30 to 40 cc. of ethjr cooled to -10*^ c., the crystals were then pressed down and 100 cc. of water coolRd to 2 or 3° C. vfRS poured through; . finally methyl alcohol cooled to -10° C. was poured through as long as any color was removed. The residue was dried and weighed. In some 56 cases the crystals were dark colored and resins appeared to be present. Kence the dried crystals vers stirred v;ith about 10 cc of ether cooled to -10° C., filtered off, washed with 10 cc. more ether, then with 5 cc. of nethyl alcohol cooled to -10° C. and finally dried and veis^hed a-^ain. The second series of washings gave parer crystals, and no further yield could be obtained from the filtrate. Cahn, Phipers, and Soam (l_7) in 1938, in briefly discussing the method of Takei and coworkers, stated that they had found the reactions involved not to be quantitative for pure rotenone and deguelin, and tliat tozicirol toheved partly. like deguelin and artificially swelled the re- sulting degj.elin contents. Jones (£8) in 1939, in using this raethod, renioved the al':<:ali- soluble material prior to the oxidation to avoid interference from toxicarol. This plan had also been used in tne v;crk of Martin and Tattersiield (84), In 1939 C-oodhij.e and Kaller (4l) developed a method for determining deguelin in derris and cube based on its raceraisation and the separation of the inactive forr. as the carbon t^^trachloride solvate. The method was as follovrs: A 50-g3i sample is extracted with chloroform in a Soxhlet for 7 hours. The chlorofom is renoved and the ejctract dis- solved in about 75 cc. of ether. This solution is extracted with two 15-cc. portions of 5-percent potassi^jn hydroxide saturated with sodium c^iloride. These portions are extracted with ether, sjid the combined ether layer is washed or.ce with 1+ 10 hydrochloric acid and once with a caturated sodium chloride solution. The alkali- soluble extract is discarded. The ether is removed, the resin dissolv'^d in 40 cc. of carbon tetrachloride, and the rotenone solvate crystalli'^ed and separated .in the uaual way. The carbon tetrachloride is removed from the . ,. • filtrate end the residue is dissolved in 10 to 15 cc. of methanol, Vrnile varm. this solution is placed in a 25-cc. Erlemneyer flask and 10 drops of 40-percent potassium hydroxide added. The contents are swirled and the flask is filled w:.th warm methanol. A one- hole stopper carrying a funnel made from a dravm-out test tube is immediately inserted so that no air bubbles remain in the flack and some of the colorless liquid is forced up into the funnel. More methanol is poured in the funnel to allow for contraction on cooling and for evaporation. The sol\itlon is kept at about 45° C. for an hour to prevent se;-a ration of resin before it is racemized, . If deguelin is oresent, crystals soon separate, but rRComization is usually not complete until the material has stood overnight. The flisk is then cooled nt 0° 3. for 1 ho^r. The icethanol is then decanted through a smell filter and the residue allowed to drain as completely as possible. For purification the degi.ielin crystals are dissolved in a little chloroform, and the chloroform is rcol?ced by evaporating to a thick solution twice vitli carbon tetrachloride, i'lially, the de.^U'Slin is crystal- liz'jo from 5 or 10 cc. of carbon c -trs-.chloride. It is usually 57 necesst-ry to S: =d the solution with the carbon tetrachldrids solvate of deguelin at 0° C. and let it stand o\'erni^ht for complete crystallization. The crystals are then filtered on a tared Gooch cracihle, vashed with cold car^bon tetrachloride saturated with deguelin, air-dried at room temperature for 4 hours, and wei^fed as the 1;1 daguelin-carbon tetrachloride solvate. The amOTint of deguelin in this iinpuro solvate is deter- mined by the red color test (40). It is assumed that deguelin alone is responsihle for the color, and the fact that racemic deguelin 5^ives only 80 percent of the color given by rotenone is taken into consideration when rotenone is us?d vs the standard of comparison. The effect of , solubility in the solvents used is comoens^.ted for by adding 0.08 percent when 5 cc. of carbon tetrachloride is us:?d for crystallization and 0.11 percent when 10 cc . is used. The accuracy of the method was checked from several angl'^s and it appeared that no great error vras introduced. The possibility of decomposition of deguelin during the racemiz^tion was studied by using a concentrated sample of active deguelin oreo^red by hi/^ .— vticuum distillation. Upon raccwizatiOR of this 'aatei-ial* 83 perdsnt of "inactive deguelin v&3 obtained. Kence not more than 17 percent was believed to be destroyed in the racemization and probably much less, as the active deguelin was not pure. The precision was said to be equal to that of the rotenone analysis (72) . Results for deguelin by this method were markedly lower than those by either the red color test (40) or the dehydro method (l20) . For example, on 13 samples of derris and cube values by racemization raaiged from less than one-tenth to about four-tenths of those by the red color test. It v;as pointed out that some of this difference mi^-t be due to compounds other than deguelin that give the color test or dehydro derivatives. As in previous work on the evaluation of rotenone-containing plants, Martin ^83) in 1940 made qiiantitative determinations of the amounts of, "deguelin concentrate" in several samples of derris. The method was .similar to that employed in the earlier investigation (84)with certain modifications. The roots vjere extracted by percolation with ethyl acet-^te for 3 hours in- stead of by prolonged extraction v;ith petroleiam ether in a Soxhlet. Alkali extraction of the resins in ether solution v;as carried out before crystal-it- lization of the rotenone. In the c-^se of roots rich in rotenone a mixture of ether and benzene was used as solvent for the extract during the alkali extraction. This scheme, which prevented crystallization of the rotenone during the extraction, has already been mentioned in connection with the determination of rotenone (59), After separation of the rotenone as the carbon tetrachloride solvate, the residual resin was designated the "deguelin concent ratei" It was also stated that an assessment of the "deguelin con- centrate" was obtained simply by subtracting from the percentage of "neutral resin" (alkali-insoluble material) the percentage of purified rotenone. •58 Discussion The Durham color reaction (75) has become established as a rapid v-qualitative test for rotenone and related compounds. Like^^dse the red dolor test originated by Gross and Smith (50^, particularly in the Goodhue modi- fication (40) . is widely used in the quantitative evaluation of material containing these substances. Since both reactions probably depend essentially on the same structural grouping, the substances present in derris and cube which v.lll" give one of these color reactions will probably give both. The dehydrd reaction has not been used so v/idely as the color tests. Both the dehydro' reaction and the color tests have been suggested as means of evaluat- ing the tftxicity of rotenone-containing plants. At one time it was thought that rotenone and deguelin were the only substances r^resent in derris and cube which were determined by these reactions, but there is now increasing evidence that this is not strictly true. Thus, althou€;h Haller and LaJorge (55), ''.'orsley ( 132 ) , Tattersfield and Martin (l20) , and other invest i.^at or s obtained comparable results on many samples by the dehydro method and by the red color test, on many other samples it has been found that the methods do not agree. For example, on a series of derris roots Jones (63) obtained results th.^t ^rere substantially lower 'by the dehydro m.ethod than by the red color method, while results for cube roots were comparable. It thus appears that in many samples substances are present which do not give both reactions, or at least not to the same degree. Furthermore, nev: compounds have been found in derris which both give the red color test and form dehydro derivatives Cl1,56_, 831 The amounts present of these other compounds have been stated to be small. The recent work of Goodhue and Haller (4l) , however, presents the possibility that large amounts of substances other than rotenone and deguelin may be determined by both the red color test and the dehydro reaction. On the other hand, it is possible that the deg-jelin may not all be present in such a form as to be determined by the racemization method. The earlier work of Haller and LaForge (55) bad already indicated that not all the deguelin was present in a single form. Only further work can give a clear understanding into the cause of the con- siderable discrepancies between this and the earlier methods. In the mean- time results by any of these methods should be considered as essentially emoirical. 59 ''tOXI CAROL AKD OTHiR ALIOLI -SOLUBLE SUBSTAJniCSS In 1935 Jones, Campbell, and SnHivan (S9) determined the amount of alKali-soluble materio.l in S'^voral samples of derris and cube roots follow- ing the method used by HalliJr and La^orge (5^) in pr'--parative work. This involved extraction of an ether solution of the ertrnct with 5-percent aqueous potassium hydi-oxids until no nore material was dissolved. The allcaline layer wap acidified and extracted with eth-=r, and the extract vss evaporated and v/eir;hed. Martin and Tattersfield (£4"^^^ 1936 i;sed essentially the same method for separating alkali-soluble and ali^c.li-insoluble fractions of derris root. In one test the separation vr^s made on an extract from which the rotenone had been crystallized; in the other the whole extract w^s used. In the latter case 5-gn. samples of root were e'ttr^'cced with ether and the ether solu- tij?sa,n!scaLe. to 50 cc*,v3s Qxtracted suc.cessively with 10, 5, andScc. of S-.percent aqueous potassi-'om hydroxide. In the case of S-^jjnatra-type and Perris malaccensis extracts jorecipitates formed in the ail^^line extract. The alkaline extract of D. elliptica resin showed no precipitate and very little alkali-soluble r.at'^-rir.l. The resins from the precipitates and alkali-soluble fractions ivere recovered Dy acidification with dilute hydrochloric acid and solution in ether. The ether extracts vrere washed, dried with anhydroiia sodium sulfate, evaporated, and dried to constant weight. Methoxyl deter- minations were made ar.d the contents of "active princdplf 3," based upon the methoxj'-l content of -ocxicarol, were calculated. In one eexies of tests saturated bariua hydroxide vas substitu>d for potash. The amount of alkali-insoluble material agreed vrlth thet obtained by the use of potash, but the barium hydro::ide extraction precipitated a largezr proportion of the alkali-soluble material than did potash. The fractionat: oxi with gqupous potash was repeated using benzene instead of ether as solvent for the extract, but only a relatively small percentage of the resin v;as extracted by the alkali from the benzene extracts of the three types of derris root exajained. Potash did not effect complete separation of the potassium ,salt of toxi- carol from a benzene solution of Sumatra-type or Derris m araccensis resins, Cahn, Phipers, and 3oam ( 17) ir. 1958 described a color test for the determination of toxicarol based on the deep green color obtained with ferric chloride. "One drop (0.06 cc.) of a 5 percent aqueous solution of com.mercial ■ferric chloride (hydrf^ed) is added to 10 cc. of 'alolliol' containing an a?20unt of Derris extract approximately equivalent to 1 mg. of toxicarol; the color developed is m.atched in a Duboscq colorimeter against that from a standard solution of exactly 1 mg. of toxicarol in 10 cc. of alcohol. The approximate amount of extract to be taicen may-be det-rmined by rough visual matching, using solutions containing 5 mg. of toxicarol or its equivalent in 10 cc. of alcohol.:'. The color is permanent for quite long periods." 60 It was found that absolute or 96-percent alcohol could "be used. Pure ferric chloride offered no advantage over the commercial grade. Ferrous sulfate gave no color, while ferric alum gave only a slight color. The size of the drop was determined by the dropping pipette arbitrarily chosen; a similar drop of 3.5-percent ferric chloride solution gave a slightly weaker color, but 1 and 2 drops of the 5-percent solution gave identical colors, '.v'ithin limits, therefore, the ano-'ont of ferric chloride appeai*ed to be immaterial provided sufficient was used. The alcoholic solution could contain at least 3 percent of acetone, benzene, or ehlo reform without interfering v/ith the results. Extracts of Sumatra- tyoe roots gave a deep green ferric chloride color and were easily matched against pure toxic?rol. Hox^ever, as the rotenone content of an extract increased the ferric chloride color became more and more brown, and vith rotenone- rich extracts matching in the Duboscq colorirr.eter was v-'^ry unci?rtain. This difficulty was overcome by employing secondary standards. Thus it was found that extracts containing less than 20 percent of rotenone could be matched against Sumatra-type extracts. One particular Sumatra- type extract was accordingly taken as a subsidiary stand- ard. For extracts containing more than 20 percent of rotenone even this was not satisfactory. Ethereal solutions of such extracts were shaken '"ith 5-percent . aqueous potassium hydroxide and the phenols recovered from the alkaline layer and separated solid salts (if any) by acidification and extraction with ether. The ferric chloride value was then determined on this alkali-soluble part only. The authors proposed the term "ferric chloride value" to denote the percentage of toxicarol that the sample would contain if the intensity of color developed was due entirely to toxicarol. Although sumatrol gave a brown color with ferric chloride, this color was believed to contain a green component v;hich contributed to the total ferric chloride color. Consequently, the ferric chloride values of derris extracts were inter- preted as giving the sum of the percentage of toxicarol and sumatrol. How- ever, the yield of sumatrol isolated from derris was said to be always much less than the yield of toxicarol- Other substances giving a green color might also be present in derris, but they had not been isolated up to this time. Rowaan and Van Duuren ( lOS ) in 1936 described a quantitative method for the determination of toxicarol based on the earlier qualitative separation of this substance by Clark ( 2l) . It was as follows: The carbon tetrachloride filtrate from the rot^^none determination is freed of solvent and the residue dissolved, by warming, in 100 cc. of 95-percent ethyl alcohol. After the addition of 10 cc. of IN. sodium hydroxide the solution is boiled. On cooling, the separated sodium salt of toxicarol is filtered, washed with alcohol, dried at 100°- 105° C. , and weighed. The weight multiplied by 0.95 gives the weight of toxicarol. The ferric chloride color test of Cahn and his coworkers (r?) was used by Jones (68) in 1939 in the examination of a series of derris and cube roots. Determinations were made by the method already described including _61 the use of secondary standards where necessary. Difficulty was #ncountered in matching the colors from the cube samples, and the use of the alkali- soluble fractions of the extracts did not improve the color matching. The amount of alkali -soluble material was determined by the method previously used (69). Martin (83) in 1940 made determinations of alkali-soluble material, or of "toxicarol fraction," on several samples of derris. The method was similar to that used in previous work (84) except that extraction of the ether solution was made first with one lot of 2-percent and then with two lots of 5-percent potassium hydroxide. As mentioned under the sections on Rotenone and Deguelin, a mixture of ether with 25 percent of benzene was employed as the solvent for extracts of high-rotenone roots. It was also stated that the percentpge of "toxicarol fraction" removed by the potash could be determined from the difference in the percentages of original and "neutral" (alkali-insoluble) resins. Discussion Both the determination of alkali-soluble material and the ferric chloride color test must be considered to give only empirical values. It is probable that in Sumatra-type derris roots both these methods give reasonably close approximations of the toxicarol plus, suraatrol content. In other samples of derris and particularly in cube samples large amounts of interfering substances are undoubtedly present. Since the ferric chloride test was proposed an additional phenol, malaccol, has been isolated from derris (88) . Rowaan and Van Duuren ( lOS ) state that toxicarol has not yet been isolated from Loncho carpus , (cube), and attempts to isolate this compound from cube root in the laboratories of this Bureau have been un- successful. Nevertheless, the samples of cube examined by Jones (68) gave ferric chloride values equivalent to 1.5 to 2.8 percent of toxicarol, presumably due to other phenolic bodies, and contained 2.8 to 5.0 percent of alkali-soluble material. The method of Rowaan and Van Duuren (l08)^ based on the separation of inactive toxicarol, should be subject to less error from interfering sub- stances. However, the accuracy of the scheme may be questioned when only small amounts of toxicarol are present. MIS-CELIANEOUS COLORIMETRIC DETEILMI NATIONS Sulfuric Acid-Nitrite Color Reaction In 1899 Sillevoldt (ll4) found that his "derrid," vhich undoubtedly contained a high percentage of rotenone, gave a brown-violet color with concentrated sulfuric acid. Danckv/ortt, Budde, and Baumgarten (25) in 1934 found that sulfuric acid followed by a small amoirnt of sodium nitrite gave an intense red-violet color with rotenone. This finding sutgests that Sillevoldt 's acid contained a trace of nitrite. V-Tien the test was applied to derris root, a 0.5-gm. sample was shaken with 5 cc. of chloroform and 1 to 2 drops of the filtered extract were evaporated on a wateh glass. To 62 this residue was added 6 to 8 drops of concentrated sulfuric acid and a crystal of sodiun nitrite. The red-violet color developed in from l/2 to 5 minutes. A similr-r result was obtained when the test was carried out in the same way "but with water instead of chloroform. The color was also given by dehydrorotenone , dehydrodoguelin, and isodihydrodehydrorotenone. Toxicarol was said to give a red-brown color only. This color reaction was developed into a quantitative test by Fischer and Nitsche (2S) in 1935. The method was essentially as follows: One cubic centimeter of an acetone solution containing 0.2 to 0,5 ng. of derris resin is placed in a 25-cc. volumetric flask and evaporated to dryness. Tbe residue is cooled and dissolved in 2 cc of concentrated sulfuric acid, and 5 drops of a 1-percent solution of sodium nitrite in concentrated sulfiiric acid are added. The flask is heated on the water bath for 8 minutes, cooled, and the solution is made to volume with concentrated s-iulfuric acid. Comparison is made in a colorimeter with the color developed from a standard solution 01 rotenone. Pure rotenone gave a "permang.'=inate-like" violet, while derris resins gave a more reddish violet. Since the authors used an "absolute" colorimeter, it was not necesspry to repeat the rotenone standard. A red filter was found to be suitable in the color measurement, although a green filter was sometimes used. Acetone, benzene, and chloroform extracts a-pve equal color values. Zther extracts, however, gave erratic results. Too much nitrite Was foiond to destroy the color. Toxicarol gave a color reaction equally as strong as did rotenone but of a more reddish violet. The mixture of dehydro comoo->jnds obtained in the Takei ( ll8 ) method gave a color exactly like that of rotenone in tone and intensity. The sulfuric acid-nitrite test was modified by Meijer (85) in 1936, In this form an aqueous susioension of the material to be test'^d was treated vlth' the reag-nt. The method was designed prin^^rily for the rapid determination of total ether extract and was said to require about 20 minut.jR. It was as follows: A 1-gm. sample of derris .root is extracted by shaking in a test tube with 10 cc. of acetone for 5 minutes. One cubic centimeter of the filtered extr-^ct is diluted to 25 cc. v;ith distilled water. This milky suspension is well sh-aken, and 0.2 cc. in a dry teat tube is treated with 5 cc. of a solution of sodium nitrite in concentrated sulfuric acid containing 10 mg. of sodium nitrite per 100 cc. The addition of the sulfuric acid to the aqueous suspension caused sufficient heat to produce the maximum color. Tae violet -color obtained was measured in a step photometer with a 5300 A. filter. The results were compared with determinations m-de on samples of known total~-extract content. The re- lation between extinction coefficient at 5300 A. and percentage of ether extract followed Beer's law. It was found that the .color could also be compared visually with permanent color standards ^rerjared from cobalt chloride. The standards v/ere made by mixing in various proportions a 10- percent solution of the cobalt salt in 96-percent alcohol, a 10-percent solution of the cobalt salt in water, and 96-percent alcohol. Ifhen kept in I sealed tubes these color standards were stable for a long period. The color reaction required a strongly acid medium. Seventy percent trichloro- acetic acid instead of sulfuric also gave the color out with a more reddish shade. Acetic, oxalic, and tartaric acids gave no color. Hydrochloric acid gave only a faint pink color. Since the varif>ty of derris known in the Dutch East Indies as "Toeba woeloeng" had previously been shown to have a definite ratio of rotenone to total extract, it was suggested that this method would give approximate values for rotenone in this type of root. , joudswaard and Timraers (43) in 1937 stated that this reaction could be usrd in the inverse sense for the detection of nitrates and nitrites in sulfuric acid. Sulfuric acid that conformed to the Dutch pharmacopoeia contained sufficient nitrites to give a color with rotenone. In the usual form of the test the color developed was proportional to the rotenone present and was stable for 24 hours. The test was said to be unsuitable for the estimation of rotenone in derris root because the reaction was not specific for rotenone. Cahn, Phipers, and Boam (17) in 1938 found that the Meijer color test was given by many substances other than derris extract; consequently, the method was applicable only in the absence of interfering substances and v/hen the genuineness of the root was certain. The color was given v.rith equal intensity by rotf^none, deguelin, toxicarol, and sunatrol, as well as by many of their derivatives. All derris extracts tested w-re fo-und to give the color with about 90 percent of the intensity given by rotenone. A modification of the Meijer test was used in this work (private communication). In 1939 Jones (68) used the sulfuric acid-nitrite test in the form described by Meijer, and made color comaarisons with a rotenone standard in a Daboscq type of colorimeter without a filter. Values for the derris samples averaged about 90 percent of the extract, but those for the cube samples were lower. The test was said to give a rough estimate of the total mrterials of the rotenone type. The Rogers end. Calamari Test Rogers and Calajnari (l02) found in 1936 that in the presence of hydrochloric acid and certain phenols rotenone developed colors ranging from blue to violet-red depending upon the solvent and the phenol used. Such organic solvents as chloroform, ethylene dichloride, carbon tetrachloride, ether, alcohol, and acetone were used. Phenol, guaiacol, and thjanol reacted similarly in these solvents. Small amounts of hydrogen peroxide, and nitric acid and light exerted a marked influence in accelerating the reaction. The color was also given by certain derivatives of rotenone. Both qualitative and quantitative tests based on this color reaction were developed. Substances usually found in proprietary liquid insecticides, such as pyrethrum extract, aliphatic thiocyanates, and oil of sassafras, were said not to interfere with the tests. In the qualitative test a chlorofonp solution of the sample vras treated with a chloroform solution 64 of thymol. A mixture,. of 0.2 part of concentrated,nitrid ^acid: and 100 parts of hydrochloric acid v/as then added and the solution shaken, k "blue-green to blue color appeared in from 30 seconds to 2 minutes vrhen rotenone was present. The following quantitative test vas designed for use with colorless liquid insecticides; To 10 ml. of a chloroform solution containing from about 0.05 to 2.5 mg. of rotenone per milliliter in a glass-stoppered cylinder, add 10 ml. of a chloroform solution of thymol (lO gm. of thymol to 100 ml. of chloroform) and -2 ml. of a reagent made by adding 2.5 ml. of o-percent hydrogen peroxide to 100 ml. of concentrated hydrochloric acid. (When the sample is not in solution in a hydrocarbon base, concentrated hydrochloric-acid may be used in place of this reagent.) Agitate for 1 minute, loosen the glass stopper, and expose the- cylinder to the intense radiation of a quartz-mercury vapor lamp (minimum output 1000 microwatts per square cubic centi.-n.eter in the field of exposure). A greenish-blue color apoears in the chloroform layer in about 15 minutes. (Exposure to bright sunlight produces simil-^r results in about 3 hours.) At the end of 30 minutes compare with stand- ards containing known quantities of pure rotenone prepared simultaneously in the same w^y. If the insecticide base is refined kerosene, use an equal amount of refined keresene in preparing the standards. ' . A more rapid method making use of an acetone solution was as follows: To 5 ml. of an ecetone solution containing 0.1 to 2.0 mg. of rotenone per milliliter add 5 ml. of an acetone solution of thymol (IC gra. of thjTnol to 100 ml. of acetone), 0.1 ml.' of 3- percent hydrogen peroxide, and 5 ml. of concentrated hydrochloric acid. A reddish-violet color aooears v/ithin 30 seconds. After 1 minute place the cylinder in a water bath at about 20^ C, and at the end of 20 minutes compare with rotenone standards similarly pre-oared at the same time. If the liquid extract contained pyrethrura extract, it was recommended tnat a standard containing pyrethrum extract be prepared to match the original color of the liquid insecticide. Cahn and Boam (l4) in the same year reported failure to obtain the Rogers and Calamari qualitative test with the rapidity and intensity stated by the authors of the test. These factors were fo\ind to be markedly affect d by the nitrous acid content of the nitric acid. With a mixture of fresh nitric and hydrochloric acids the rotenone color was produced only slowly. V/hen nitrite was added to the mixture, or vrhen the acid mixture was allowed to stand before use, the rotenone color developed rapidly. It Was concluded that the reaction depended more upon the presence or production of nitrous acid than upon the presence of nitric acid. In their reply Rogers and Calamari (l03) stated that it was essential to use concentrated hydrochloric acid containing not less than 35 percent 65 of hydrogen chloride and pointed o'jt t}iat Cshn and Eoam used an acid contain- ing only atout ?0 percent of hydrogen chloride. They found that when the stronger hydrochloric acid was used the nitrogen peroxide content of the nitric p.ci'i v/as unimportant. The intensity and speed of the color reaction depended on the concentration of the acid used. They "believed that the acid reag. nt depended on the formation of nitrosyl chloride for its action and that the reagent should stand at least 3 minutes "before use. Cahn and Boam (l5) in 1937 reported that when they used 3.-6.45 petcent hydrochloric acid (sp. gr. 1.18) their results were in su"bstantial agreement wit"n those of Rogers and CalaiTari. They found, hovrever, that even t'nen the rate of development of color nnd its final intensity increased greatly as the acid mixture was allowed to stand "before use. They emphasized that the sensitivity of the reaction depended .rreatly on the exact conditions used. According to DeOng (27) the Rogers and Calamari method proved un- satisfactory for the quantitative det ormination of rotenone in fly sprays. In 1339 Jcnes (68) modified the Rogerr, and Calamari test for the analysis of several s;amoles of derris and cu"be roots. The color produced in the acetone test was not proportional to the rotenone present. In chloroform solution the proportionality "between color and concentration held. Eeat as well as light accelerated the color formation, "but the latter v-as adopted. The solutions v/ere exposed in glass cylinders to daylight (not direct sunlig'nt) for PA hours. Perchloric acid ufas found to effect a more rapid development of the color than hydrogen peroxide. At a rotenone concentration of 0.12 mg. per cubic centimeter, 2 drops of 60-percent perchloric acid gr've a moderately intense, pure-"blue color in 24 "noiirs, Zven in this form results v/ere erratic and duplicate standards varied as much as 10 percent. Deguelin was found to give a color intensity a"bout 125 percent of that given "by rotenone, whereas toxicarol gave only a"bout one-balf the rotenone color. VJhen rotenone was used as a standard values for the derris roo Us ranged from 60 to 120 percent, and those for cube and tim"bo from 140 to 160 percent of the total extractives. The method of Rogers pnd Calamari v/as recently used as a qualitative test for rotenone in miscellaneous insecticides (127) • The characteristic "blue color did not develop vrhen too much rotenone was present; therefore, in case of failure to obtain the test a repeat should be made with a smaller sample. Other Color Reactions Geoffrey (29) in 1B95 was the first to study the color reactions of rotenone with several reagents. The most characteristic involved treatment with bromine, followed 'by application of concentrated sulfuric acid to the residue. A violet color was produced, Dennis (26), in a patent on cube issued in 1927, described a color reaction for testing the material using sulfuric and nitric acids followed by potasuum hydroxide. Jones (6S) in 1939 found that this te:.t gave identic?! color reactions with roots of derris, cube, and Tephrosia virgiriana. 66 Schmitt ( l09 ) in 1930 stpted that rotenone may be detected in ground root "by treatment vrith nitric acid, v;hereapon the affected portions turned red. Pozzi-~scot (95) in 1936 stated that rotenone dissolved in concentrated sulfuric acid with the formation of a red-cerise or rose- cerise coIoj:. When the acid contained mercuric oxide in solution, as in Denige's reagent, the color reaction vrith rotenone was from 100 to 1,000 times more sensitive and was not subject to interft;rence from as many other substances as vrhen acid alone was used. On being warmed with this reagent, the rotenone disBolved giving an intense orange-yellow color. The intensity of color t\'as observed to be proportional to the amount of rotenone. In later work Pozzi-iscot (98 ) discussed the color reactions given by sulfuric acid, sulfuric and nitric acids, and Denige's reagent ^^dth sub- stances that might interfere with the rotenone reaction, and described methods for differentiating between the reaction of rotenone and reactions of other substances. In 1937 Tapia Treses ( ll9 ) reported that rotenone gave a red-violet color with a solution containing 0.1 gm. of van?»dium pentoxide in 10 cc of sulfuric acid. OTHSH T)ETIHMINATIO!TS Polarimetric Methods Danckwortt and Budde (?4) in 1933 stated that polarim?tric determinat- ions might ultimately be used for the evaluation of derris root. In the same year Jones (62) briefly studied the possibility of using the high optical rotation of rotenone (74) (in benzene [alpha]r^= ^24° for a 5-percent solution) as a means for its determination. The optical rotatory powers of derris and cube extracts were det'^rmined in several solvents, and a hypothetical rotenone content was calculated from the values for pure rotenone. In most cases the results v;^.^re very much higher than those obtained by crystallization. Since one derris extract was dextrorotat- ory, it was concluded that optical rotation cannot be used as a measure of the amount of rotenone present in the root. A polarimetric method proposed by Danckwortt, Eudde, and Baumgarten (25) in 1934 was claimed to be superior to the crystallization methods, at least as a measure of the toxicity of derris. Directions were as follows: 3 gm. of finely pulverized sample is digested with 30 cc. of benzene at room temperature for ?4 hours. The optical rotation of the filtered benzene solution is determined in a 100-mm. tube. Tae rotenone content is obtained by the follow- ing formula: Percent rotenone - alp^ui X 1.000 233 In 10 out of 11 samples of derris root the method gave values higher than those by cr^'stallization. The method was also adapted to the deter- 57 .. mlnation of rcten.one in aq-j^iouf? extracts of derris, ji.nd this use of the procedure was describ'^d in det.-i.il. Gstirner (5l) in 1934 emp] o/ed the method of Dnnckvortt and covrorkers in the anal;ysi5 of 19 sniiiples ar.d fractions of sanples of derria root. In all cases e:ccept on© results ivere higher than those by crystallization, in most casus jnarkedly hjguor. This diffe-^tnce was attributed to the rotenone remaining in tiio. rosin from t.hs crystallization. Lanckv/ortt' B m'^thod -ms also us^d by Fischer and Titcche (28) in 1935 in a study of chemical methods for the evaluation of commercial derris preparations. These Investigators found, that extraction of the sample for this jjurposewas comolete in 3 to 4 hours. In certain cases it was necessary to have more derris resin prec-sont than specified by lanckvortt and coworkers. Rowann (l05) in 1935 report''-^d that in analyse'? of samples of derris root the rotenone coxitent vsras usually highor by the polarimetric method of Danckwortt and his cov/orkers tV.an by crystallisation, but that in two samples the values by crystallization were abort double tncse by the optical- rotation method. Eo-^aan also reported the isolation of a dextrorotatory preparation of toxicarol from derris. because of these circumstances analysts were warned against the use of the polarimetric method. In reply Danckwortt (.2Z) pointed out that the polari-^etric method Was. a means of determining tiie effective value of derri? and not of deter- mining rotenone. Ee also reported the use of the method in a study of the relative strbillty of inductrial derris extracts ever a. piriod of time. The extracts wore dried V'ith smd, the residue was shairen vdth benzene for 20 minutes, ana the optical rotation of the solution was ifttermined as previously described. Stror^gly colored solutions were shaken with animal charcoal. Too much ch^^rcoal was avoided, since it caused a decrease in optical activity. Rowaan (106) in 1936 again warned against the use of the polarimetric method for the determination of rotenone in derris rnd stated that the most reliable method was soma modification of the extraction-crystallization method, Tattersfield and Martin (i;23.) in 1S36 studied the optical activity of benzene solutions of resiis and v,heir fractions derived from samples of Der ris elliptic a. D. roalac ^ ccnsis , and Sumatr-'^-t^/pe derris root. A fraction of Sumatra-type fesin obtained oy means of methyl alcohol was dextrorotatory in methyl alcohol but levorotatory in benzene, A sttidy of the optical rotatory povr~r of acetone and benzone extracts of 15 saonples of derris and 10 samples of cube root was reported by Jones (65) in 1936. Calculations of hj-pothetical rotenene contents wore based on the kr.own rotations of pure rotenone in acetone and bonzene, as was done in earlier work (62). All extracts were levorotatory in benzene, but 68 acetone extracts of certain derris roots with little or no rotenone by crystallisation were dextrorotatory. There vas no agreement oetween the value obtained by crystallization and the values obtained from the optical rotatory powers of either acetone or benzene extr-'-cts. A further calculation based on optical rotatory poP©r which used the rotations of both acetone and benzene extracts wa« nade by Jones. It was based on the assuT.ption that the optical rotatory power of all the rotenonelike com-..our.ds oresent was greater in benzene than in acetone to the same degree as ifas that of rotenone, and that the rotation of the oth r optically active material \-/as the same in both solvents. All values by this method were "marlcedly higher thao values obtainedb crystallization. It was concluded that the ootical rotatory pover could not be rccomiTiended as a means of evalu.?tion of derris and cube root. One of the laboratories particioating in the collaborative analysis of derris report«^d by Koolha--?s and Meijer (see p. 35 J in 1937 used the oolari- metric method of Danckwortt, The use of this method for the determination of rotenone was criticized by the authors of the report because of the general lacs of knowledge of the significanc3 of optical activity and of the propor- tion, of other constituents present. Vorsley (l32) in 1937 studied the use of optical rotatory pow-ir as a means of assessing the toxicity of dorris root. The dritrd cixtract from hot percolr-tion of the root with cthj'-l acetste Was dissolved in benzene and the concentration of the solution adjust, d so that the angle of rotation in a 200-mm, tube was about )22 degrees. Concentrations bet'A'.^cn 25 and 200 gm of original sample in 25 cc. of benzene were involved. Direct extraction with benzene in a Sorhlet gave the sajnc results. From a curve for the optical rotation of pure rotenone a value was read off corresponding to the angle obtained as described in 'Vorsley's method for rotenone .dctermin?.tion (l30). Because in most samples this value agreed rather closely ^-rith th-?t for dehydro compounds, Worsley used the term "optical dohydro conipo^onds." In a subsequent article Worsley (l33) proposed the term "optical constituent?^ " instead of "optical dehydro compounds" for the value described in the previous work. He also proposed the use of the reciprocal of this value for toxicity como;^risons. Guillaurae and Procschel (54) in 1937 st-^ted that the polarimetric method was usad in CJermany, but that investigators in other countries found it gnve too high r: suits. Rowapn and Van Duuren ( 108 ) in 1938 confirmed in general the observations of Jones (65 ) on the optical activity of derris and cube extracts, particular- ly with regard to the dextrorotation of derris extracts that gave no rotenone by crystallization . Thung (123) in 1939 stated tha.t in the analysis of derris powder a 2- gm. sample was extracted by shaking repeatedly with 53 cc. of benzene during 24 hours. The optical rotation of the filtered extract was determined in a 20CLmm, tube (0.5^ V. • l percent rotenone). 69 Determination of Methoxyl Content Tattersfield and Roach (l22) in 1923 st.^tud that tlie genuineness of derris eX-t?rcts night b,e Gmfirm-^d "by the datcrmin-'tion of their methoxyl ccntont. Dctprainations w^rs Tr.gde on the dried ether .ext recti ves by Perkin' b modification (91) of the Zeisol !.r,_ethod. The methcicyl content of different samples of e-k'tract ran,'^ -d between 15.5 and 14.7 p<2rcent. ' ■ ' Dimci^'ortt end 'Bw^'rle (p.-^) in 1933 doterr.iincd the me^hoxyl contont'of benzr-^ne- extracts of dc-rris root. In 1?Z4: DEncjC\'ortt., Buddp, p.nd Eaijjng?rt<^n. (25) described their method for the deterjiinstion of the mcthcjcyl content of derris extract. The benzene extrp.ctvas' 'transferred directly to the flask of a Zeisel methoxyl determins-.- tion apparatus and evjiT'Orat^d to dryness therein. The ueual Zeisel method was used. One gr^^n of £;ilv?r iodiae correspor.ded to 0-6394 ^ram of "active substance," calculptod as rotenone. This det: rmin:^ oion was said to have only an orienting vrTae^ Campboll, Sullivan, and Jr-cs '.18) in iS'Sl.det'^rrmin^d the raethoxyyl contents of derris and cube roots by tho method of Yieb'^iclc and Schwappach as modified by Clerk' (S?-) •, The detcrminetion.'^s w^^re itade on acetone ertractives, which had been tr^afd with benzone to rcs'ove tr^^ces of acetone. In 1935 these invesoi^ators (69) used the S8"?ie method on benzeno e:rtracts of derris and cube and calculat.t^d the results to nat:;rial having the same raethoxyl content as rotenone. These values v/ere said to be significant, as all the kno'.vn naturally occvrri::^- compounds of the rotenone group had approximately the saTie methoxyl ccntonc as rotenone, Methoxyl determinations •■-ere made by Tattersfield and Martin (l20) in 1935 on samples cf three s-oecies of ^^tZli* Values were determined on dried ether extracts by the method of Cl^^rir Cp.o) <, The results were closely correlotea vith the weig.its of the e:;her extract. Martin and Tattersfield (84) in 1936 fcmd the methoxyl content of the ether extrects of thrc-e samples of derris root in good a;^Teement with that of the benzene extrasis. They also mr;de msthoxyl deter-ni nations on various fractions of the extracts. G-uillavjne and Eerv^ (55) in 1939 used a modification of the Zeisel method in det-^-rmining tha methoxyl content of derris, cube, and other rotenone - containing materials. The m.othyl iodide forraed was ran into OclU silver nitrate and the excr:ss of silver nitrate vras titrated vdth 0,1 I\f sodiiom thiosulfgto. The vpluo obtained w^s calculated to m.aterial of tho sane methox;rl content as rotenone. The results, an the 11 samples tested, were in good agreem.ent vdth those by the modified Durham blue color test used by these investigators, Kiscellaneoua Determinations Danckvrortt and a^dde {PA) in 1953 stated th^t attempts to measure the absorption of iodine or bro-m.ine by the double bond of rotenone as a method 0^ determination l.:^d to practical difficulties. Again in l':^54 they reported (25) that, since the halogen coTiSumption was dependent on factors other than 70 the quantity of rotenone, a reproducible method could not "be attained, Vhittaker and G-lickman (129) in 1934 described a method for the detormination of rotenone which was an adaptation of the method of Gnadinger and Corl (39) for the pyrethrins. It involved reduction of an alkaline copper solution, a modified Folin's solution, "by standard solutions of dextrose and of rotenone and "by the unknown solution of rotenoae. The reagents and pro- cedure' were almost identical with those described by G-nadinger and Corl. A standard rotenone solution in 95-percent ethyl alcohol was used. Results for standard roterojao solutions rang^^d from 9.60 mg. of rotenone equivalent in reducing action to 1.25 mg. of dextrose to 21.09 mg. of rotenone equiva- lent to 4.19 mg, of dextrose. Prom these results an equation was obtained for calculating the amount of rotenone present from the equivalent amount of dextrose. In a second series of tests the results agreed well with those calculated from this equation. The precision of the method was said to be usually about 2 parts per thousand. . Traces of chloroform and carbon tetrachloride interfered with the determination. This procedure was successfully used for the determination of rotenone in alcoholic solutions containing antioxidants. 'tJhen the method was applied to the analysis of derris root, the results vere much higher than those obtained by the crystallization method. For example, two samples which gave about 6 percent of rotenone by crystallization gave 11.5 and IS^-S pcrcant by the reduction method, A sample with no rotenone gave 15.5 percent by the present method. - 71 - LIKRA.TU3E CITSD 1. Af^ROSE, A. M, , pud H.AA.a, H. B. 1936, Toxicolofrical study of derria, Indus, pnd En.-^in. Chom, 28:315-3^1. 2. ASSOCIATION OF OFFICIAL AGFilCULTU.^VL Ck^MlSTS. 1940. Official arid tentative method^ of analysis of tho Associa- tion of Official Agrlc^jilt^iral Chemists, Ed. 5, 757 pp., illus, 3. JBEACH, I). C. 1936, Hotenoao detenrdnation - observations on the dotonnina* tioa of rotenone v.dth a sia£-,g;ested method. Soap 12(7): 109,111-112. 4. £2GI;PJJF, F. L. 1937, Bot3non"bc3l/ennielsc i Dorrin-og OcloT'^dAcT, Dansk. Tidsskr, Farra, 11: 6-12. 5. SSGUE. H, 1939, ' Lg do«age chimiquo des poudros rotenonecs. Ann. Agron, [Paris] 9: 121-132. 6. HERTAUD-BOSSI 1938, La rotenone ses proprietoE et gcs applications. Hun. Colon, Ann. (19o8) 2: 1-139. 7. iiIiA.CK.IE, W. J. 1S32.. A new apparatus for trie continuous extraction of plant materials with ether under tropical conditions. Soc, Chem, Ind. Jcur. (Trans, and Commun. ) 51: 129-130. 8. 19:,3. Dorris uligin osa. Agr. Jour. (Fiji) 5(1); 34-35, 9, BBJL^K, E. R, 1939, Evaluation of dorris and allied inr.ecticides. The Kg ther land 3- Indies 7(8): 4-8, 10. BP.Ovn^E, C. A., and SKIM-EH, V,'. ^^. 1931. Wiley's Principles pJid practice of agricultural analysis, 2. . Fertilizers and insecticides. 1.6, 3, 645 pp. 11, BUCKLE2, T. A. 1935, The tozic constituents of derris root, Soc. Chem, Ind, Jour. (Trans, and Goraaun. ) 55: 235-291. 12. CAHH, R." S. ' ■ ■ 1935, Derris versus war'ble. Fanner and Stock-Breeder and Ai;r. C-az, 50: 333, illus. - 72 - • 13.. GABK4H,'^,aJ^'^ BCA;^, '^. J... 1935. Detersinat.ibn of rotenone in dcrris root and re sir.. Soc. Chena. Ind, C'cr-iv, (Trans, and Co-ninun. ) 54: 37-42, 14, and 2a?e!, J. J. 1935, Coloar roaction for rotenone, Soc. Chem. Ind, Jo-JLr, (Ch.cn, and Ind.) 55; 384, 15, 4. ... • and 30j^, J. J. 1937, Colour test for rotenone, Soc, Chem. Ind, Jour, (Chen, -pnd Ind.) 55: Si- 22, 16, and iiOAM, J, J, ' 1933, The approximate detcnination of rotenonss in dorris, Soc. Chea, Ind. Jour, (vrans. and Coiniaun, ) 58: 194-196. 17, , PHIPEP..3. R, ?. , find 2'X^K, J. J. 1938, The touti conposition of Pcrris extract, Soc. Chom, Ind, . . -Jour, (Tr&xa, and Commju. ) 57: 2C0-209, 18, GAI'IPBELL. F. L. , SULLI7a;I, v. JJ. , fj..^ J'ol3:3, H. A. 1934. Derris in fly gprays. Kerosene extracts of derris root as . house fly sprats, ITothod and results of laboratory tests of extracts of derris and cuoe rootc;. Part I. Soap 10(3): 81-33, 85, 87, 103, 105, 107. 19, CASSIL, C. C. 1941. Terris residue on laarlccta'ble ca'D'bai'^e, Jour. Econ. Ent, 34: 7P-74, 20, COA'ALIER, J., pud CE3TALISR, M. 1957, Les plantcs a rotenone: derris, cube, tinbo. Bui. dcs Sci, Pharmacol. 44-: 223-241. 21, CUFJT, E. P. • . 1930, To3d carol, A constitu.?nt of the South Anerican fish poison Cracca ( Tenhrosia ) toxicarig. . Jour, Aaor, Chem. Soc, 52: 2461-24'54. 22. 1933. The ViebScl^ and Schv.-appach method for the determination of mothozyl and cthoiQ^l groups. Jour, Assoc. Off. Agr, Chem. .15: 136-140. 23, DAJ^CKWORTT, P. V/, 1935, Untorsuchungen von DerrisprSparaten. II. Uber die IVortbo- stimmung von Derriswurz-.ln und die Kaltbarkeit dos Kotenons in Hand elspra"oara ten. Arch, der Pharm. 273: 385- 388, - 73 24. aUICKTjr^KTT , P. tv.. Mid BTJUDE, E. ' 1933, Uebor die WGrfbestimmimg dor Dcrrisv/urzol. Deiit, Tierarztl, 'ichnschr. 41(^3): 677. 25. : , , BUDDE, H., and BAW''GARTSN, G. 1954. Pie Worfbo^timnung von Derrisvurzcln. Arch, dor -^hsmi, 272: 561-559. ' 26. DEIaCIS, W. J. 1927. Vermifuge and insecticide, U, S. "^atont 1,621,240, issuod ^^a^ch 15. 27. m, ONO, E. H." 1938. ny- spray analysis. Soap 14(10): 91, 93, 95. 28. FISCIEiE, W., and 1.1TSCII3, &. 1935, ^iGthoden zur Prlifung von -^flanzonschutzmitteln. IX. Die 3raucliT3arkoit ciniger Schnollmcthodcn zur chemischon Pnifung von Dorris-oxtraiiten und ihr Vergleich mit dcr Diologiscixcn Prufuiig dorsorocn Zxtrakte an Kiofom- und Soidonspinn>;^rra.upen. Biol. Roichsanet. f . Land v^ lorstw. (Mitt.) oO: 57-78. 29. GSOPMOY, E. 1895, Contribution a I'otude du Hopinia nicou Aublet au point de vue botaniq.uo, ciiimiquo ct physio logiq.ue. Marseille Inst, Colon, Ann. (1895) 2: 1-86, illus. 30. G30RCtI, C, D, V, 1934, Division of chemistry; Annual report for the year 1933. ^cd. l^alaj/ States, Dept. Agr. [£ul.] Gen. Scv.. 19: 17-24. 31. «* 1957. A new laothod of harvesting, drying and sampling derris root. Malayan Agr. 'Jour. 25: 425-429. 32. and CURTLflR, S. A, 1929. The periodic harvesting of tu'oa root ( Derris elliptica Senth. ). I!alayan Agr. Jour. 17: 326-334. 33. and TEIK, G, L. 1932. The rotenono contant of Malayan tuba root, Malayan Agr, Jour. 20:. 49 8-507. 34. pJid TSIK, G. L, 1933, The valuation of tuba root. Fed. Malay States Dopt, Agr, [Bui.] Sci. Ser, 12, 30 pp. 35. and TEIK, G. L. 1936,. UotGs on the preparation of derris root for export together with a suggested method for evaluation, ^"^alayan Agr, Jour. 24: 489-502. - 74 - 36. G-20SGI, C. E. V., and TEIK, G. L. ... 1937, Hote on the. estimation of rotenone in derri.s root. -Malayan Agr. Jour, 25: 23c 37. and T?IK, G, .L. 1939, ?rel.viT;_aary' rrcults cf analrsis of clonal types of derris ' rcAer r.lclc. co'^di L-ioas, ^^alayaii Agr, Oovrc 27: CCi-.Zol, 38. Gi::iiE^TTE, J, ;o, 1923. Mal-7 ->ol sons and charm cures, 3d. 2, 260 pp., illus, L-j.'iaoii, 39. GilADTHG.^R. G„ B, ,^ and GORIj, 0, S. 1939. Sc .CJ.cj. :i! pr jochrum flowcr.s, I, The quantitative detorrnina- Uir»:,. ••:/ iij^ r.ccivo principles. Jour. Ame r„ GhcE. Soc, bit ?CL^i--3C'S4. 40., GGCOaU3, L,. 'D. 1936, An xraproTement, on the Gross and Smith colorJ metric mcfiod fjr *v,"uD do'i c?^".inat: on of rote-'oxic ar.d deguslin. Jcur, Assoc, Off. Agr. Chom. 19: 118-120. ^tl, and HALjjIIE., H. L. 1939. A mothcd for determining dcguelin in derris and cube. Indus, and Sngin. Chem., -^nal. £d. 11: 640-642. 42. snd eall;:;s, h, l. 1940. nctcction and estimation of dihydro rotenone in the hydro- gonai'ion pr.)dv.'.'vs cf rotenone. Indus, .and j^ngin. Chem., Anal. M. 1.5? 652--q54. and PROSSGI-iEL, A, ■ 1937, Etudes do plantos a rotonone: precedes de dosage. Rev, de i^ot, Appl. ot d'Agr. Trop, 17: 737-743, 55. HALL3F.,' H, L. , and La?0EG3j F. B. 1934i, Rotenono, 11^^ '^'he non-crystalline constituents of derris root. Jour. Amer. Chem. Soc. 56: 2415-2419. 56. HA5PZR, S, H. 1939. The active principles of leguminous fish-poison plants, • Part II „ The isolation of 1-eliiptone from Derris elllptlca . Jour. Chem. Soc, 1939: 1099-1105, 57. HOLIviAiJ, H, J, 1940. A s\irvsy of insecticide ;materials of vegctaDle origin, • 155 pp. Imp' rial Institute, London, 5,8.. . IICPZRIAL liMSvITUTE 1938.- Recent research on empire products. [Gr. -orit.j Imp, Inst, Sul, 36(4): 527-529. 1939. Survey of collal:orative work on the analysis of derris root carried out TDetwoen the Imperial Institute and Rotharasted Experiment Station, 11 pp., mimeo. - 76 - 60. ISHIKaWA, T. 1916, Investigation of tulDa, an East Indian' poison for fish, Tokyo Igakkwai Zasshi 30: 45-46. Japan. Med. Lit. 1 (pt.2): 7-8. [In Japanese. Abstract in Chom. Abs. 11: 2370. 1917.] 61. JOiJSS, H. A. 1931. Carton tetrachloride may replace ether in the extraction of rotenone. Preliminary report. Indus, and Sngin. Chem. News Ed. 9: 301. 63, 64. 65. 66. 67. 68. 1S33. Assay of plant material for its rotenone content. An ex- traction method. Indus, and Engin. Chem., Anal. Ed, 5: 2&-26. 1933. The rotenone content of derris root, cube root and other ■olant materials. Jour, Wash. Acad. Sci. 23: 36-4t6, 1933, Notes on the occurr-wncc of rotenone in species of Derris and Loncho carpus. Jour. Wash. Acad. Sci. 23: 493-496, 1936, The optical rotatory power of extracts of derris and cube roots. Jour. Agr. Res. 53: 831-839. 1937. Determination of rotenone in derris and cube. Crystalliza- tion from extracts. Indus, and Engin. Chem., Anal, Ed. 9; 206-rao, 1933, A titrimetric step in determining rotenone. Indus, and Engin. Chem., Anal. Ed, 10: 684-685. 1939. Colorimetric evaluation of derris and cube roots. Indus, and Engin. Chem,, Anal. Ed. 11: 429-431. 69. ■ — , CAIIPBELL, I, L., and SULLIVAx", W. N. 1935. Relations between chemical composition and insecticidal effectiveness of rotenono-b earing plants. Jour, Econ, Ent. 28: 285-292. 70. ■ , C.^i-BELL, f, L. , and SULLIVAl^i, W. N. It35. Cracca - a source of insecticides; A preliminary study of domestic species of devil's shoestring as sources of insecticidal materials. Soap 11(9): 99, 101. 103, 105, 107. 109. . -i 77 ~ _^ . 71. JQi^rS^liA srd C^MKA^, J.- J. Ti ■ ■ ...... 19'6B, Determination of i-ot<3nono in df>rris r.nd cube, II. Sxtraction from the root, Indus. anc5 ."^r.gir.. Chen., Anal. Ed. 10: •19-^23, ,■;,'. 72. and GEARaM, -J. -J, T. . 1936, DeterK.l.'ii-.tion of rotonone in deri*i 9 end cuIdo, III. An im- proved ciystalli'^atlon iB&tho/l» Jour, Assoc^ Off^ AtSr. ■' Cheui., 21: 148- ■ 151, ' 73^ PjidLOVE, S. • 1907. The solulixity of ro^Gnone„ II. Data for certp.in additional solvents, Jcur. AiuQv, Cxiein. Soc. 59: ,26e4-r:;S96, 74, -— and SIIITH, 0. M. 1930, The coluMlity of rotenone. I. Solnbilit/ arid optical rotation in certain orga;.:ic solvents at 20'^. Jcur. Amer. Chem, Eoc, 52; 255'x~3562, 75, and 3>'ITH, C. l\ 1933, A color te^it- for rotenone, Indus, and £n;^in, . Chem., Anal. Ed. 5: 75-76. ' 76. — .. and SULLIV.UT, W. N, 1938. Evalv.ating derrir. and cube: 'Iho question of to'tal -extractive content, Joxir. Econ. Int, '31: 400-405, 77. KOLOriAAL IKSTITUUT T3 A-ISTSHT.M 1950, Toe"ba-vorcel. Inlichtingen en oi..derzoelc-lngen- v&n d-e s.ideellng- hrndels'niu.seuun i'n 19^.9, Aiusborda^l Kolon, Inst., Afd. Eas.ders'iras. 8, Feded. 26: 31-99, illus. 78. KOOLEAAS, T. R. , , . -- 1932. The analypAs of derria roots and the estimation of the rotenone -content, Buit-enzorg Jardin Bot. Bui, 12J 533-574. 79. ■■ 'and l-IEIJER, T, M. - ^ ■• ■•■"".'.. 1933, Eigenschappen van wortels van versch-iHende Cerris-'soortcn, Bergcultures 12: 10=5-1051. 80. KRUKOiT, B. A., and SlilTH, A. C. .. . , 1937. ■ Ro t onone-yi 3 Idint: plants of South i^jnefica. Airer. Jour, ■ ■ •■ ■Bot,-'24: .573-587. . . 81. KUIiAG^VA. M. , and SUTO, K, . . ' ■ • ,' . 1908. Einh'^ues Verfaliren-^ur quahtitativon Eectiamung des Eettes tmd der unverseifbaren Subctanzen in tierischen Material nebst. der Kritik einiger gebrauchlicben i^ethoden. I. >> . Biochem, Ztschr, 8: 212-347.- . - 78 - 82. LSVALLOIS, J, 1937, Obserrations sur Ics insecticides rotSnones. Compt. ILend, 17th Cong. Chim. Ind., Paris: 559-561. 83. I'JlETIN, J. T. 1940, The prohlom of the evaluation of rotenone- containing plants, V, The relative toxicities of different species of Derris . Ann, Appl. Biol, 27: 274-294, 84. and TATTSBSilSID, F. 1936, ^'he pro'blem of the evaluation of rotenone-containing plants. II, Derris elliptic?- . Derris malaccensis and tha "Suioatra- type" roots. Ann, Appl. Biol, 23: 880-898. 85. irSIJZR, T, H, 1936, Approximate colorimetric determination of derris extract, Rec. des Trav. Chim. dos Pays-Bas 55i 954-958,' Also, Eenvoudige colorimstrische methode oa ongevser het Gxtractgehalte van Derriswortel te bopalen. Sorgcultures 10: 1169-1170. 86, 87, 1337, Over de waardeering van derris, -^uitenzorg (Java) Experiment Station, Heport of 25th Meeting, October 1937, pp. 181-194. 1938, Senige eigenschappon van derriewortel, Bcr^TCultures 12: 1562-1563, 88. and KOOLHA-^iS. D. R. 1939, Kew constituents of derris root. I, Rec. des Trav. Chim, des Pays-Bas 58: 207-217, 89. and KOCL'^AAS, D. R, 1940, Determination of rotenone in derris root, Indus, fiid -^ngin. Chem,, Anal. Ed., 12: 205-209. 90. "JAGaI, K, 1902. [Uber Rotenon, ein wirksamcr Bestandteil dor Derriswarzel,] Jour. Tokyo Chem. See, 23: 740, [in Japanese. Reviewed in Biochem. Ztschr, 157: 2. 1925, j 91. FJEKIN, W. E, 1903, Simplification of Zeisol's method of methoxyl and ethoxyl determinations, [LondonJ Chem. Soc. Jour. 83: 1367. 92. PEYil. W, ajid HiilTORBEIlT, E, 1931. Ueber Derris elliptica . Apothokei^Zoit. 46: 1435-1488. 93. P0Z2-I-ESCOT, E. 1935, Dosage de la rotenont dans les veg^taux du genre Derris . Ann. de Chim. et Analyt. 17; 233-235. 95. 97. 93, • •:** 79 ** -OZZI-SSCCT^ E» .18354 ..I-odificacion al procedimiento do dosada de la; rotenona. Soc. ^m* Peru ^oU 1(4): 50, 1953. Investigacioneg sobre las reaociones de la rotenona (II parte). Hev. Cien. 38('*17): 31-25* lb-36. .La inyestigacion iflicroqxiimica de la rotenona, Sev, Cien. 38(^18): 63-64. 1S37, imevas indicaciones para el dosado de la rotenona en los vecetales. Hev. Cien. 38(4?0): 41-46. 1937, , Rotenona, III. Parte,- Investigacionv^s sobre las reacciones sulfuricas, sulfo-mercuricas y de Durham. Rev, Cien. 38(420): 47-51. 99. si:-:"jRir, j. . 1^";37, De I'emploi du pyrethre et du derris dans la lutte centre les ensectes, oournees de la lutte chimique centre les ennemis des cultures, Paris, "'ay, p. 6|^-69. [Review cd oy Guillaujne and Herv', Rev, de 5ot. Appl. ct d'Agr. Trop. 19: 552-564 ,(1939)]. 100.. ROAri, R. C. • 1930. The Araericaja market for tuba root ( Derris •olliptica ). ■'alayan Agr. Jour, 18: 455-458. 101. . ROBIITSOIT, L, A. 1S3S, IvQte on the estimation of rotenone in British G-uiana hpiari Brit. Guiana Agr. Jour. 7: 191-192. 102. RO&iRS, n. D., and GAlAiJlRI, J. A. " " " 193$, Rotenone determination by colorimetric methods, Indus, and ing. Chem. , Anal. 3d. 8: .135.. 103. and CAJ4A:,-iRI, J. D. -1936. Colour reaction for rotenone. Soc. Chem, Indus, Jour, (Chem. and Indus.) 55: 788, 104. RO'i.A-ni;;, F. A, 1935. De chemische waardebopaling v?n rotenonhoudend planten- materiaal (derris-wortel, loncho carpus- vortcl, enz.), Chem. iveekblad. 32: 291-295. 105. 1935. Die isestimiaung des Rotenongehaltos von Derriswurzoln. Arch, der Pharm, 275: 237-258. ■^ . A 80 ^ 106. .... 1935. DetspaliniW^ja to tenon in derris-wortel. 'Cheia. /eek"blad. 33: 9.- ' ■ 107, 109. 112, 1$37. Rotenon'bepaling in derriswortel. Cheni. 'Yeekblad. 34: 605-606. lOS. ?.nd VA1>T DUTZSl.', A. C, 1958. Over de analyse van derris - en lonchocarpuswortels en da ' ■ • ' spjnsnstelling van bun .extracten. Ghom. 'veek'blad 35: 755-756, .'5r:-""T'?i? 1?30. I'erris elliptica Bentii. , ein vcgetablischer iind ungiftiger Ins^cticidlieferant, Angew. -^ot. 1.2: 453-463. 110. SCHOJTBiH:-, S. 1938. Deterinination colori;aetrique da la rotcnone. Compt, Rend, 17.tn Cong, Chim. In(i., Paris:' 947-952. 111. SEASZR, 7f. :•. 1937. ITotes on the determination of rotonone. Soc. Chem. Ind, Jour. (Trans, and Oorniun. ) 56: 168-173.. ' " 1933. Sone further examples of rotonone determinations on derris, timbo, and "bar^oasco. Soc. Chem, Ind. Jour. (Trans, and Gommun. ) 57: 372. 113. SIDViaS, A. 1,, PUSS2LL, 0. A., LOvrL-U^, K, S. , '?0?;lic;E, 2. D.,' ' £SLA_'S0i:,'-C. 0., and LlTTLi, V. a. K-38. Studies' on the possibilities of devil's shoestring ( Teohrosia virgiiii. ana ) rJid other native species of Tephrosia as co.nimercial sources df insecticides. U. S. Dcpt. Agr. ,'Tech, 3ul. 595, 40 pp., illus. ll-i! SILI2:V0LDT, H. .2. T. VAIT 1899. Uebcr das Derrid' und ?a,chyrhizid; eiii Beitrag zur Kenntnis der indisdheh Pischgifte. Arch, der Pharm. 237: 595-S16. " '• 115. SPOOi^I, I. ♦•;. , ' ■ ', •1931. ''Vaamemihgen over de samenstelling van derris— "''ortel uit xTod. Oost-I'ndie,' in "verb and met sijnc eventuelle warrde als insecticide. Ser. .\fdool. Handelsmusouun, Koninkr. Ver. Amsterdam Xolon. Inst. ^!0, 63; also in Iridischc "ercuur 54: 351-355, 115, nnd xvC*/A.-vi'I, p. A, l&oo. Gronc" stof f en voor het insecticide rotenon in Nod. Oost-en '..'ost-Indio. Bor. .A.fdeel, Pl^ndclsmuseuurn KoninkrVt^r. Amstordnm Kolon. Inst. -:o. 79; also in Indische ^ ercuur 56: 321-523. -si- ll?. TAK.EI, S. 1923. The constituents of derris root. I. Inst. Phys, and Chem. Res., Japan, Bul^ 2: 485-495. [in Japanese. A^bstract in Cham, Abs. 13: 685, 1924. j 118. .. MIYAJiyj^, 3., and ONO. M. 1933. Uber Px)tenon, den wiirksnmen ^estajidteil der EGrriswiirzel, XI. Rotenonharz. quantitative Bestimraung des Rotenons und des DcguOlins im Botenonharz. Deut. Chen. &esell. ier. 66: 1826-1333. 119. TaPIA FHSS3S, A. 1937, Reacciones de la quinina y de la rotcnona con el reactivo vanadico. Sec. ^ui-:i, Peru i^ol. 3: 219-220. 120. TATTilRSFISLD, ?y snd I^JlBTII:, J. T, 1935. The problem of the evn.luation of rotenone-containing plants. I, lerris 'jlliotica and Derris aalaccensis . Ann, .-ippl. 3iol, 22: 576-605. 121. and MASTI!!, J. T. 1936, '^he problem of the eval^'ortion of rotenone-containinr:; plants, III. A study of the o-otical activities of the resins of D, ellj-pticp . D. gq.l?c;censi3 and the "Suiaatra-tjrpo" roots. i.nn. Appl. Biol. 25: 399-bl6. 122. — and ROACH, W, A. 1923. 1'he chemical properties of Derris elliptica (tuba root), Ann. At5pl. Eiol. 10: 1-17, 123. THUi-i&. T. H. 1939. Phytopathologische waarnemingen. •'"roefsta, vorstenl. Tabak, ileded. 87: 23-46. 124. UlTITSD STATiiS DSPART^ '.'IZivT OJ AGPJCULTURS, AGPJ CULTl?HAL IIARKETIL'G SIRVICE , 1939. Detemination of ether soluble extractive material in aerris or cube povc^ier in presence of sulfur. U. S. Dept. Agr. , .-igr. i'-^arket. Ser"/. , Insecticide Div., ^-ethod Ijo. 751,1, 1 p. , ;niraeo. 125. 126. 1939, (qualitative test for rotenone in mineral oil fly sprays, U.S. Dept. Agr., Agr. I'arKet, Serv. , Insecticide Div,, , 1-iethod i'o, 752, 1 p., mi.i^eo. 1939, Determination of rotenone in derris and cube povi'der in presence of sulfur. U.S. Dept. Agr., Agr. Market. Serv., Insecticide liv, , i'lethod ilo, 753.1, 2 pp., mimeo. UNIVERSITY OF FLORIDA 131. 132, 133. - 82 3 1262 09224 7757 127, UlUTSD STATES DSPAIffiCSKT 0? AG2I CULrJ:RE, A(>51 CULTUPAL MUQCSTING SrSTICS. 1941. "qualitative test for rotenone. U. S. Pept. Agr. , Agr. Mai^cet. Serv. , Insecticide Biv. , liethod to. 752.1, 1 p., typswritten. 123. . Fj^RTO RICO Z.XPS"itIIiLJT STATIOiT Eeport of the PuSrto Eico Experiment Station 1940, In pre3S. 129. WEITTL^JGIB, H. II., p^d G-LICICiAiT, I. 193^, The oxidation of roteione "by copper in an alkaline acdiiim. Rcc. des Trav. Ciiim. des ?ays-3as 53: 1145-1150. 130. VORSli'^A', R. E. LeG, 1933. Eooenone, Part I. The detsrrdnation of rotenone. Soc. Chea. Ind. Jour. (Trans, rjid Conmun. ) 55: 349-357. 1937, Ths inse-'ticici -il properties "of some East African plants, III. -'-.du?. ^-1 suDr-.o ea ^snth. Part 2. Cher.ic£:,l const i-.-.^ents, Ann. Ar^pl. ^iol. 24: 651-658. 1937. Iiotenone- Part II. Evaluation of plants containing rotenone, Soc. Chea. Ind, Jour. (Trans, and Coitinran. ) 56: 15-23, 1937. The eval^ar-ticn of Perr-is and ^^i^n^/JLlea. Soc. Chem. I (Trans, and Gomraun, ) 56: 175-175. nd. Jour. J