1357
V O L U M E 2 4 , NO. 8, A U G U S T 1 9 5 2 ( 2 ) Higeleisen, .J.. and Kant, .I.. unpublished data.
carry-over effect. K O carr>--over w:td observed in similar tePts made with ammonia and phosphine. The mass spectrometer was flushed with a sample of gas to be analyzed if the sample differed markedly from the previous one. The time required for convtarsiori of R $ample was 15 t o 30 niinutes. The conversion of propune required that the uranium be maintained at a temperature higher than that needed for water or ammonia. At 400' C. with propane the met.al became coated with a protective lager which inhil)itcri reaction not only with that gas but with many othcrs also. Trwtnient tTith nitric acid restored t,he activity of the metal.
(3) Chenouard, J., Geuron, J.. and Roth, E., with technical collaboration of Paoli, O., and Lecomte, J., Commission for
Atomic Energy, France, Rept. 8 7 , 1951. (4) Friedman, L., and Irsa, .I.P., ASAL. CHEM.,2 4 , 8 7 6 (1952). (5) Giaff, J . , and Rittenberg. D.. I b i d . , 2 4 , 878 (1952). ( 6 ) Kamen, M.D., "Radioactive Tracem in Biology," p. 131, Sew (7)
York, Academic Press, 1948. Kirshenbaum, l., "Physical Properties and Analysis of Heavy Water," Chap. 4, Section 5 , Ne-iv Tork, hicGraw-Hill Book
co., 1951
(8) Xewton, -4.. 8., Manhattan District, Declassified Docitment 724 (9)
(January 1947). Sier, A. O., Inghram, M. G., Stevens, C . .i.,and Rustad, B., Ibid.. 197 (1947).
(10) Orchin, & I . , Wender, I., and Friedel, R.
-I., ANAL.CHEX..2 1 ,
1072 (1949).
( 1 1 ) Sprinson, D. B., and Rittenberg, D., C-. (Supplement), 115, S9 (1948). RECEIVED for review February 6, 195?.
S.. T a d M e d . Bu12.
.iccepted . i p r i I 29, 1962.
Estimation of Pyrethrins on Coated Paper Bags FRED I. EDW-ARDS
AND
CIPRIANO CUETO
Bureau of Err torrroloz?. arid Plant Quarantine, U . S. Department of Agriculture, Beltsville, .Wd.
RESEST interest in the stoinge of foodstuffs in paper bags 'that have been treated Kith pyrethrum has necessitated the development of an analytical procedure for estimating the content of p-yrethrins in these bag coatings. both for control in nianufacture and for following the stal>ilit!- of the pyrethrins during s t n i q e of the bags.
2 3 Cm. LONG 0.0. 7rnm., FLARE, 0.D. I5mm., 4 cm. LONG
DISTRIBUTOR TUBE,
30cm.
-,-
/SIDE
'---BARREL,O.D. Figure 1.
A R M O.D. 15mm.
28mm.
F:xtraction ippiratus
Lord (a) has described a color reaction applicable t o the pyrethrins which has been adapted t o the estimation of the pyrethrin content of these paper bag coatings. The basic steps of this procedure are: reaction of pyrethrins and hydroxylamine to form a hydroxmic acid, formation of a colored complex by reac-
tion of the hvdroxaniic acid LT ith ferric chloride. and evaluation of thiq colored coniplev in a colorinieter PREPARATION O F STASDARD CURVE
Reagents. PTRETHRUJC COXCESTR.\TE.Select a 20"; concentrate which contains a pyrethrin I to pyrethrin I1 ratio of 1.16 f 0.1 to 1 [based on analysis following the method described in ( 1 ) . The pretreatment x i t h cold Skellysolve F must be carefully folloa-ed before analysis in all cases] , STAh-DARD S O L U T I O S , 0.1000 gram Of pyrethrins (based 011 f ) in 1000 ml. of et)hyl alcohol. 1 ml. = 0.1 m y . of pyrethrins. Hydrosylamine hydrochloride, 2 .If aqueous solution, to he made u p frefih weekly. Sodium hydroxide, 3.5 2V aqueouy solution. Hydrochloric acid, 4.0 S. Ferric chloride (c.P. anhydrous sublimed). 0.37 -\I i i i 0.1 S hydrochloric acid, t o be made u p fresh s-eekl!.. Ethyl alcohol, 95%. Skellysolve F, boiling point 30-60" c'. Sodium chloride, ACS grade. HvAo Super-Cel, a diatomaceous filtering aid. Apparatus. Nett-Summerson photoelectric colorimeter, Model 900-3, with 12.5-nim. calibrated test tubes and green filter having a transmittance peak a t 540 mp (any comparable coloi,iriieter may be used). Extraction apparatus, see Figure 1. Procedure. Aliquots of the standard aolution of pyrethrin3 are pipetted into 500-ml. separat,ory funnels; 5, 10, 15, 20, 25, and 30 ml., equivalent t o 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg. of pyrethrins, are used. Ethyl alcohol k added to each separatory funnel to bring the total volume t o 50 ml. Then 350 nil. of water. 15 ml. of Skellysolve F, and 7 grams of sodium chloride are added. The solution is shaken for 1 minute and allowed to separate, and the aqueous layer is transferred to a second separator) funnel. Ten milliliters of Skellysolve F is added to the second funnel, and the solution is again estractrd for 1 minute. Thir: extraction is repeated in a third wparatory funnel and th(, a q u e ous layer is then discarded. The three Skellysolve F extracts are combined, a minimum of olve F being used for the transfer. The conibined shed once with 5 ml. of water and filtered into a 50ml. volumetric flask through a small plug of cotton wet with Skellysolve F. Sufficient Skellysolve F is used in the transfer to bring the total volume to 45 nil. The Skellysolve F is then evaporated by immersing the flask in a water bath at 25' C . and passing a stream of nit,rogen into the flask. Three milliliters of ethyl alcohol are added and thp flask is swirled t o dissolve the residue. Six milliliters of alkaline h3-droxylamine solution (prepared within 30 minutes of use by mixing equal volumes of 2 h!J hydroxylamine hydrochloride solution and 3.5 N sodium hydroxide solution) are added and the flask is alloxved t o stand with intermittent shaking for esactly 10 minutes at 25" Ilt 5" C. At the end of this 10-minute period 3 ml. of 4.0 N hydrochloric acid solution are added, the flask is shaken thoroughly, and 3 ml. of the ferric chloride solution are added,
ANALYTICAL CHEMISTRY
1358 bringing the total volume to 15 nil. The flask is shaken again to effect mixing. Then 0.2 to 0.4 gram of Hyflo Super-Cel is added and mixed with the solution by vigorous shaking. The solution is filtered through a 12.5-cm. folded filter paper directly into the photometer test tube. Since the color intensity declines gradually with time (0.13% per minute), the time of addition of the ferric chloride reagent is noted, and the transmittance reading is taken exactly 10 minutes later. A reagent blank is carried through wit,h the standards under exactly the same conditions and is used to set the photometer a t 100% transmittance. Since the Klett-Sumnieruon colorimeter is equipped with a logarithmic scale, in draxirig the standard curve, concentration iri milligrams of pyret,hrins per 15 ml. of solution is plotted against, the dial reading. For other instruments not so equipped, coilcentration is plotted against log l / T . Straight-line curves should he obtained. ESTIklATION OF PYRETHRINS OY COATED PAPER
Reagents and Apparatus. Essentially the same as those used for preparation of standard curve. Procedure. The final solution for analysis should contain 1 to 3 nig. of pyrethrins. A quantity of paper that will contain approximately this amount in its coating is selected and cut into strips 6 inches long and 0.28 inch wide. These strips are placed upright in the barrel of the extraction apparatus. The apparatus is assembled, and 95% ethyl alcohol is added to fill the barrel to the side arm and approximately half of the flask, to which several glass beads have been added. The barrel is hrated by immersing it in a water bath at 80" to 85' C. The flask is heated hy placing it in a bath of Wood's metal or oil, the temperature of n-hich is regulated so that vigorous but not violent boiling occurs (100" to 120' C.). The heating is continued for 3 hours, and the extractor. is then disconnected and the solution in the flask is concentrated to ahout 28 ml. by continued heating in the bath. The contents of the flask are transferred t,o a 500-ml. separatory funnel with sufficient alcohol to bring the total volume to 50 ml. The analysis is then continued as in the preparation of the standard curve. .Ireagent blank is run with the samples for analysis under identical ronditions and is used t o set the colorimeter a t 100yotransmitt,ance. Where it is possible a check analysis on untreated kraft paper of t,he same origin is recommended. DISCUSSION
Eight samples were run to determine t,he effect of varying times of reaction after the addition of the alkaline hydroxylamine reagent. Five milliliters of a solution of purified pyrethrum in Skellysolve F were evaporated t o dryness and taken up in 3 mi. of 95% ethyl alcohol, and 6 ml. of alkaline hydroxylamine solution were added. Precisely timed intervals of reaction were used, the reaction being stopped by the addition of 3 ml. of the hydrochloric acid reagent. Transmittance readings were made exactly 10 minutes after the addition of ferric chloride reagent. T h r results of thesr tests are shown in Table I. Based upon these data ii reaction time of 10 minutes was chosen as an optimal point. To determine the effect of temperature on the course and spced of the hydroxylamine reaction, replicate samples were run a t three temperatures using a 10-minute reaction time in t h r hydrosylaminr reaction and in the ferric chloride rcaction. The rwults of this series arc shon n in Tahlc I1 ~~
.~~
Table I. Sample N o .
~~~~
~~~~~~
~
Effect of Reaction Time Reaction Time, hlinutes 1 0 5 0 5 0
Klett Units 135 165
175 175
7 0 10 0
175 178
30 0 60 0 90 0
184
18%
Table 11. Effect of Temperature Tetnn.,
C.
Klett reading
18 184 183 183
34 183 181 180
.52 1fis
163 170
From these results it was concluded that a temperature of 25 f 5' C. would give opt,imal accuracy and reproducibility. The question of p H was not exhaustively investigated. I t can be stated, however, that the solution should be definitely acidic at the time of addition of ferric chloride reagent. The various normalities of the reagents have been calculated to give these conditions and should be carefully observed. During the devclopmental work on this method some variation of the optical density was found to be due to differences in the ferric chloride used. One sample gave a deep coloration of the blank and another caused discoloration and fading of the blank. This difficulty was eliminated when c.P., anhydrous, sublimed ferric chloride was used in preparing the reagent. Since the ratio of pyrethrin I to pyrethrin I1 is variable and since Lord ( 2 ) stated that the color developed by pyrethrin I1 is more intense than that developed by pyrethrin I, the effect of this variation in pyrethrin I to pyrethrin I1 ratio was investigated. The records of commercial preparations analyzed by this laboratory show a pyrethrin I to pyrethrin I1 ratio of 1.16 i 0.16 to 1. Accordingly, samples ;1and B, representing, respectively. the niaximum and minimum pyrethrin I cont,ent, were selected for investigation to determine thr error which could be introduced hy this variation.
Table 111. Effect of Varying Ratio of Pyrethrin I to Pyrethrin I1 Sample S o . 1 2 3
Klett Reuding per Mg. of PyrethrinsSample 4 Sample B 71 92 i3 93 72 43
The resultant data (Table 111)sho4 ed that the maximum error which could be introduced by these variations in pyrethrin I content when a sample is selected a t random, within the limits 1.16 + 0.16 to 1, would be 14%. However, since the mean pyrethrin I to pyrethrin I1 ratio (1.16 & 0.1 to 1 ) is called for in the preparation of the standard curve, the maximum error encountered \vi11 be half that stated above-that is, 7y0, If a sample of the original niaterial used in treating the paper Iiags is av:iiiable, this may be used to prepare the standard curve. If this is done the error due to varying pyrethrin I content is eliminated and the met,hod n-ill have ii niaximum experimental error of 1.5% (calculated from the results given in Tahle I11 using either 5ample -2 or R. The errnr hetn.wii Rlcatt readings on replicate3 is experimental error). Thus, three procedurcs are available, each with a definite drgree of precision: Standard selected at random within the rat,io 1.16 i 0.16 to 1 for pyrethrin I to pyrethrin I1 (maximum error of 14%) Standard selected to conform to mean ratio of pyrethrin I to pyrethrin I1 (1.16 i 0.1 to 1) (maximum error of 7 % ) Standard prepared using original coating material (maximum errnr of 1.5%) The procedure sclccted \vi11 naturally depend on both the material available for the preparation of the standard rurve, and the desired precision. % . major difficulty \vas encountered in the extraction of the pyrethrins from the paper. r\fter various extraction methods had been tried, the procedure described was found to give satisfactory results. Results of eleven tests of this extraction on paper coated in this laboratory with known amounts of pyrethrins indicated a recovery ranging from 9 i to 100 and averaging 98yo,. Because it was possible that the coating applied in the laboratory was not m firmly applied as a commercial coating, a series of extractions n-as run t u determine the completeness of est'raction in the specified time of 3 hours. Recovery n-as considered com-
V O L U M E 24, NO. 8, A U G U S T 1 9 5 2 Table 11’.
1359
It will be noted that the decrease in the amount of apparent pyrethrins found on the coated paper is as great as 80%. Coated papers 1 and 2 in Table IV were of unknonn origin and no history was available other than that they were early experimental production. Ho\rever, coated paper 3 was of recent manufacture and was coated by the manufacturer to contain approximately 6 nig. per square foot. The results of analysis using the Skellysolve F wash averaged 5.8 mg per square foot, which is consistent with the manufacturer’s claim. The results of analysis v ithout this IT ash averaged 23.0 mg per square foot and indicate the presence of large amounts of interfering substances which must be removed. The uncoated paper still shows a slight color development even after a Skellysolve F extraction. Although this value iq small, it is desirable to run a control analysis on untreated kr:ift paper of the same origin as the treated bags whenever possible
Effect of Skellysolve F Extraction .4pparent Pyrethrins, 11g. N o extraction Extraction
Pyrethrum concentra.te Purified Crude (20%) Coated paper KO. 1 XO. 2 No. 3 Untreated paper
so.1 so. 2
1.26 2.57
1,25 1.97
2 . .53 3 83 23.0
0 49 i.75 J 8
1.05
0.15 0.09
0 71
plcte, since continued extraction of the samples for periods as high as 6 hours showed no additional pj-rethrins when the extracsts were analyzed. Because continuous heating of the alcoholic solution of pyrethrins might be causing loss through thermal deconiposition, a series of tests was run using aliquots of a standard solution of pyrethrins. One series of aliquots was analyzed directly without heating and the second and third series were placed in extraction apparatus and heated for 3 and 6 hours, respectively. Subsequent analysis shon-ctl no cpintitative difference in color developed in these three series. The alcoholic extract of thc papor is r\tract,ed with Skellysolve F in the preparation of thp sample for color development in order to separat,e thc pyrethrins fmm insecticidally inert related conipounds which are extracted from the paper by alcohol but are not soluble in Skellysolve F. This Skcllysolve F extraction is further necessitated by a similar soluliilit,y of other esters which \yere found in t8hekraft paper and sizing. The results obtained with and without this Skellysol~eF extraction are sh0n.n in Table I\-.
ACKNOWLEDG.VlENT
The authors are indebted to Elmer E. Fleck and to Paul A. Giang, of the Bureau of Entomology and Plant Quarantine, the former for many helpful suggestions, and the latter for X.O.A.C. analyses. LITERATURE CITED
(1) Assoc. Offic. Agr. Chemists, “Methods of .Inalysis,” 7th e d . ,
Method 5 : 114, Washington, D. C., 1950. ( 2 ) Lord, K . il., Sature, 165, KO.4197, 567-8 (1950). RECEIVED for review February 27, 1951. Accepted M a y 8, 1952. Presented before the Division of Agricultural and Food Chemistry at t h e 121st \feeting of the AMERICAN CHEMICAL SOCIETY,Milwaukee. \Tis.
An Anomaly in the Zeisel Methoxy Determination R . L. HUANG AND FRANCIS MORSINGH Cnirersity of Malaya, Sinfiapore, Malaya
lII1 Zeisel method L generallj- regarded as specific for oxygroups, carbon-methyl groups usually being ge:-methyl determined by the Kuhn-Roth method. While engaged in a search for new estrogenic substance3, the authors synthesized the diphenol (I, Table I j and found, rather surprisingly, that though devoid of methoxy groups. it nevertheless gave a small but, definite value for “methoxy” using the Zeisel method. Siniilar substances (11) and (111 have been prepared and found to behave in the same n-aj.: the valuer of methoxy found, though not constant, correspond to roughly one quarter of a inole of methyl iodide being formed from one mole of suhstance analyzed
(see Table I). It is noteworthy that the trialkyl derivative (Y),synthesized and analyzed some years ago (3),did not, give any methoxy under the same conditions of analysis, while the value found for the dimethoq- derivative (IV) agreed crith t,he calculated value. Hence, it appears that this behavior is peculiar to the fully methylated dibenzL-Is such as (1) and constitutes an instance in ivhich the specificity of the Zeisel methosy determination is questionahle. [a similar anomaly has been observed in D-mannitol ( I ) . ] The effect of tetra-alkylation hy g r o u p other than methyl might be 15-orth study. Compound (1111 was prepared by the method of Farmer and Moore ( 2 ) ; the synthesis of ( I ) , hy three different methods, and of (11) arid (IY)will he published elsewhere. Results of Zeisel llethouy Determinations on Various Compounds ~~
Tahle I.
.kCKZIOWLEDGMENT \-/
Substance
--Analy&_
-_
c, 56
Calcd. 2,3-l)iinethyl-2,3-di (p-hydroxyphenyl) butane (I)
50.0
2,3-Diinethyl-2,3-di (p-nitrophenyllhutane (11) 2,3-Dimethyl-2..3-diphenylhiitane (111) 2,3-Dim~thyI-2.3-di(p-methoxypheny1)hutane iI V) 2->Iethyl-2,3-di (p-hydroxyp1ienyl)pentane i V )
6.i.8
Found 80.0 6.5 6
H, % -~ Calcd. Found 8.15 8.21 6.10
\-/
1
_ ~_ _~.-
s,%
Calcd.
Found
..
~~~~
O l l e . “c Calrd. Found 0 3.10, 3.76, 4.32 0 1.88
5 94
8.53
8 20
..
..
..
.
..
20.8
..
0
80.3
80.2
8.79
8.74
80 0
75.6
8 15
8.00
,
0
3.33
20.4 0
The authors thank the Shell Co., Ltd., for a fellon-shill awarded to Francis Morsingh. Microanalyses were carried out by If-eiler and Strauss of Oxford, England. LITERATURE CITED
*X.raki,T., a n d Ilasi, T., J . C h e n ~ hoc. . J a p a n , 61, 99 (1940) ( 2 ) Farmer, E. H., a n d Moore, C . G., J .
(1)
Chem. SOC.,1951, 141. ( 3 ) Huang, R. L., Ph.D. thesis, University of Oxford, England, 1 9 4 i . RECEIVED f o r review Fehruary 21, 1932. cepted May 1 , l(152.
Ac-
