Natural Pest Control Agents

6 Analysis of Pyrethrins HERMAN BECKMAN and PAUL T. ALLEN

1

Downloaded by CORNELL UNIV on August 17, 2016 | http://pubs.acs.org Publication Date: January 1, 1966 | doi: 10.1021/ba-1966-0053.ch006

Agricultural Toxicology and Residue Research Laboratory, University of California, Davis, Calif.

The development and adaptation of procedures for the separation, isolation, purification, identification, and analysis of the components of the pyrethrum mixture have been studied and evaluated. Results of studies to determine the molar extinction coefficient of pyrethrin I as well as a gas chromatographic procedure for the determination of pyrethrins are reported. The use of chromatographic separation procedures (including partition, adsorption, gas, and thin-layer chromatography), colorimetry, and infrared spectrophotometry are discussed.

j p o r several years this laboratory has taken an active interest i n the

analysis o f p y r e t h r u m preparations a n d i m p r o v e d procedures ( 7 , 2 ) . T h e aim of the present investigation was to develop a p r o cedure, based o n gas chromatography, that w o u l d quantitatively measure the f o u r principal constituents of the p y r e t h r u m mixture. Progress has been made t o w a r d this goal. A t this point i n our study w e have many facts and the first step t o w a r d the complete method.

Crosby; Natural Pest Control Agents Advances in Chemistry; American Chemical Society: Washington, DC, 1966.

52

NATURAL

CH \ / C CHr-C=CH

PEST CONTROL

AGENTS

5

I I ™ H-C—C=0 Η

3

0=C-0-CH

3

PYRETHRIN CH Downloaded by CORNELL UNIV on August 17, 2016 | http://pubs.acs.org Publication Date: January 1, 1966 | doi: 10.1021/ba-1966-0053.ch006

y

m

C>v

.CH« CH

C H - C — C-C-O-CH V \ CH £ CH. H - C, — C=0 o \^C J i CH C-C=CH Η CH 3

\CH

3

3

CINERIN

C

H-C—C=0

CH3-C=CH 0=C-0-CH

I

Η 3

CINERIN

I

It is apparent f r o m the literature o n p y r e t h r u m analysis that there is little agreement o n what is needed or wanted i n n e w method ology. A s soon as i t is possible to obtain complete information o n all the significant components o f the p y r e t h r u m mixture, a better under standing o f the methodology needed w i l l be available. T h e purpose o f this paper, then, is to summarize the present condition o f some o f the w o r k done i n this and other laboratories and present information o n the separation, isolation, and identification o f some o f the components o f p y r e t h r u m . W o r k of others has, i n part, been confirmed and some n e w evidence is presented o n compounds isolated f r o m the p y r e t h r u m mixture. Separation

Procédures

C o l u m n Chromatography. L i q u i d - l i q u i d partitioning has been used b y several investigators f o r several pesticides, i n c l u d i n g p y r e t h r u m (1,2,3,5,10,13). T h e most useful procedure allows the


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A N D ALLEN

53

Analysis of Pyrethrins

separation of the bulk of the n o n p y r e t h r o i d extractives f r o m the active constitutents

of the pyrethrins.

P r o b a b l y the t w o most c o m m o n

solvent pairs are nitromethane w i t h hexane hexane.

and acetonitrile

with

D i m e t h y l s u l f o x i d e and dimethylformamide have been

ex

perimented w i t h o n a small scale as the immiscible phase w i t h hexane. N o conclusive information has been developed as to their potential. Partition c o l u m n chromatography f o r separating several of the p r i m a r y constituents of the p y r e t h r u m extract

has been reported.


T h e elution pattern f o r some of the constituents of the p y r e t h r u m mixture recovered f r o m the partition c o l u m n is s h o w n i n T a b l e I. Table I . Elution Order of Pyrethrum Constituents from Gas Chromatograph as Compared with That from Partition Column Identifications i n terms of gas chromatographic responses, infrared data, and colorimetric tests Elution Order Compound Cinerin I Cinerin I type Pyrethrin I Cinerin II type Cinerin II Pyrethrin II

B.P.,°C.(9)

Gas

Partition

chromatograph

column

1 2 3 4 5 6

1 2 3 5 4 6

136-38°/8X10- mm. 3

146-48°/2X10- mm. 3

182-84°/lX10~ mm. 196-98 7 7 X 1 0 " m m . 3

3

T h e elution pattern f r o m the gas chromatograph is i n c l u d e d f o r comparison, as w e l l as the boiling points f o r the k n o w n compounds as given b y E l l i o t t ( 7 ) . peaks.

F i g u r e 1 identifies the gas chromatographic

In general, a silicic acid c o l u m n is used as the supporting

medium f o r acetonitrile or nitromethane, w i t h hexane as the mobile phase. Some w o r k using Celite i n place of silicic acid has been done to fractionate the p y r e t h r u m components nitromethane-hexane

(Figures 5 and 6 ) .

The

solvent mixture was used w i t h a silicic acid

c o l u m n as the basis f o r separations i n a colorimetric analysis f o r pyrethrins (1).

In this procedure p y r e t h r i n I was recovered i n quan

tity and used to relate to the total amount present. T h e colorimetric procedure w i l l detect compounds containing conjugated

unsatura-

tion i n the side chain. T h e side chain must also terminate i n a double bond as w i t h pyrethrins I and II.

T h u s , no color is observed w i t h



54

NATURAL

PEST CONTROL

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ο

TIME, MINUTES

RATE, 2 MIN./ INCH

Figure 2. Gas chromatographic responses from a pyrethrum concentrate Identity of numbered peaks given in text

Figure 2. Infrared spectrum of methyl-trans-chrysanthemate Prepared from DL-trans-chrysanthemum acid and collected as pure ester from gas chromatograph



6.

B E C K M A N AND ALLEN


55

Figure 3. Infrared spectrum of methyl-cis-chrysanthemate Prepared from DL-cis-chrysanthemum acid and collected as pure ester from gas chromatograph

Figure 4. Infrared spectrum of methyl-trans-pyrethrate Prepared from pyrethric acid isolated from hydrolysis mixture of pyrethrum concentrate. Pure ester isolated by gas chromatography



56

N A T U R A L PEST CONTROL AGENTS

Figure 5. Infrared spectrum of pyrethrin II Fraction isolated from silicic acid-acetonitrile-hexane partition column

cinerin I or II or related compounds such as allethrin. T h e recently reported jasmolin I (7) w i l l not respond to the color test. A d s o r p t i o n c o l u m n chromatography has been employed to sep arate the constituents of p y r e t h r u m . Florisil and aluminum oxide have been used as adsorption columns to retain m u c h of the p i g mented materials. T h e pyrethroids m a y be caused to elute w i t h several solvents. In our experience mixtures of hexane w i t h ethyl acetate, methanol, ethyl ether, dichloromethane, or acetone have p r o vided different elution patterns. Gas Chromatography. In recent w o r k w e have f o l l o w e d the progress of a partition c o l u m n using a combination of thermal c o n ductivity gas chromatography and the colorimetric test. T h i s has shown that some of the unidentified peaks originally observed b y gas chromatography and not identified are related to the general p y r e t h r u m structure. These compounds are present as part of the original mixture as they have been separated b y the c o l u m n partition chromatography and give the same infrared spectra before and after gas chromatography. T h e i r retention times correspond to peaks f o u n d in the mixture and are judged not to be thermally isomerized products as has been suggested (3,4,6,1).



6.

BECKMAN

AND

ALLEN


Figure 6. Infrared spectrum of cinerin II Collected from gas chromatograph after isolation from silicic acid-acetonitrile-hexane partition column

Figure 7. Infrared spectrum of pyrethrin I Isolated after dual partition chromatography First partition column. Celite-acetonitrile-hexane Second partition column. Silicic acid-nitromethane-hexane (with 5% acetone). Corresponds to peak 3 of gas chromatographic separation of pyrethrum mixture


57

58



Colorimetric

Analysis

The colorimetric procedure has been applied to each of the frac tions isolated from the partition column. T h e response to the color test has allowed an accurate prediction of the general type of infrared spectra ultimately found. T h e color test has also been applied to fractions collected from the gas chromatograph. O f the major responses observed when the pyrethrum mixture is passed through the chromatograph, three of the components respond to the color test. A t least two other pyrethrin-like compounds of long retention and small quantity also give the color test. N o infrared data are available on these. While the colorimetric procedure has not found wide applica tion as a quantitative method, it has proved its worth as a diagnostic tool. The separations allowed by the partition column provided a rather pure sample of pyrethrin I, demonstrated by the gas chro matograph and by comparison with known infrared spectra. T h e puri fied pyrethrin I was weighed quantitatively and a color test performed to determine the extinction coefficient. The figure obtained from ten runs is 1120, calculated from the formula: A a = — be where A is the absorbance measured, b is the path length of the cell in centimeters, and c is the molar concentration of the compound. Under the conditions of operation we observed the following: 0.25 a= 1 X (0.232 Χ 1 0 " ) 1120 4

a=

This is expressed as molar absorptivity at 550 χημ. T h i n - L a y e r Chromatography. A study using thin-layer chro matography both for separating components of a pyrethrum extract and for purifying single components has been made. Other reports have mentioned the use of thin-layer chromatography in various con nections related to pyrethrin analysis. Stahl (14) reported on pyre thrins I and II and evidence for presence of other compounds from


6.

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AND ALLEN


59


p y r e t h r u m concentrate. Part of the additional compounds f o u n d were the result of ultraviolet irradiation of the p y r e t h r u m mixture. T h i n - l a y e r plates were made w i t h silica g e l - c a l c i u m sulfate and each contained a mixture of zinc silicate and zinc c a d m i u m sulfide as phosphors. Separated components are generally visible under ultra violet light b y fluorescence quenching. T h i s was true, i n part, f o r the pyrethrins, except that some of the separated components possessed a natural fluorescence under the ultraviolet lamps.

Figure 8. Infrared spectrum of a cinerin I type Fraction isolated from Celite-acetonitrile-hexane partition column and rechromatographed on a silicic acid-nitromethane-hexane (with 5 % acetone) partition column. Gas chromatography indicates purity of 98% A solvent system consisting of hexane-ethyl acetate-benzene (75-15-10) was f o u n d the most appropriate f o r resolving the c o m ponents of p y r e t h r u m . W h e n a p y r e t h r u m concentrate was spotted and developed, a series of separated components was observed. T h e observation was made possible b y using a v i e w i n g cabinet fitted w i t h l o n g - and short-wavelength ultraviolet lamps. F r o m seven to nine constituents were observed, depending o n w h i c h lamp was used. A l l of the same components were not necessarily observed w i t h each lamp. O n e of the most interesting observations was that of a brilliant red spot w i t h an Rf of about 0.85, w h i c h was observable o n l y under the long-wavelength lamp. T h e spot was bright and tight even at a


60



relatively high Rf value. T h e c o m p o u n d has not been identified. It is not one of the k n o w n p y r e t h r i n or cinerin compounds, however. T h e multiplicity of responses makes thin-layer chromatography not particularly suited f o r p y r e t h r u m analysis, either qualitative or quantitative. It d i d c o n f i r m , however, that the crude oleoresin c o n tains several p y r e t h r o i d compounds i n substantial quantity, as previ ously s h o w n b y gas chromatography w o r k . T h i n - l a y e r chromatography was also used i n an attempt further to p u r i f y fractions that had been separated b y c o l u m n or gas c h r o matography. Rather precise Rf values f o r several compounds were

Figure 9. Infrared spectrum of pyrethrin I decomposition product isolated from thin-layer chromatographic plate determined, but complications were injected into the total separation picture. W h e n e v e r , f o r example, p y r e t h r i n I was spotted o n a plate and the chromatogram developed, t w o spots were observed: the original material and a decomposition product of higher Rf (see Figure 9 ) . T h e decomposition product rechromatographed w i t h o u t change, but the recovered p y r e t h r i n I o n rechromatography again showed further decomposition. It was determined that this degrada tion took place i n air and was not caused b y ultraviolet radiation. Quantitative

Gas Chromatographic

Method

O n e purpose of this w o r k was to develop a gas chromatographic procedure based o n separation and quantitation of each o f the c o m -


6.

61


B E C K M A N AND ALLEN


ponents k n o w n to be present i n the mixture. T h i s rather ambitious goal has not yet been reached. A s an o u t g r o w t h of this w o r k , h o w ever, a quantitative analytical method has been developed, based o n gas chromatography. T h e procedure starts w i t h the A O A C (8,11) hydrolysis to y i e l d the chrysanthemic and p y r e t h r i c acids. T h e acids are recovered, esterified w i t h diazomethane, and determined b y gas chromatography. Since p y r e t h r i n I and cinerin I give the same monocarboxylic acid, and p y r e t h r i n II and cinerin II give the same dicarboxylic acid, quantitation is based o n the same criteria as the A O A C method. It is n o w suspected that other components of the CH 0 CH»-C C-C-OCH, \ / 0 C κ ι C-C=CH ο CH 3

ÇH 0 CH.-C C-C-OCH, \ / V Ç CH C=CH 3

3

r

CH

3

CH

3

3

METHYL CHRYSANTHEMATE DIMETHYL PYRETHRATE mixture contribute to the total monocarboxylic and dicarboxylic acids f o u n d . Table II. Determination of Total Pyrethrin I and Cinerin I Sample No.

AOAC

1 2

11.38 6.1 11.38 6.1 9.83 11.38 11.81 10.98 9.97 11.38 11.38 9.83 11.81

3

4 5

6

7 8 9 10 11 12 13

% Recovery,

%Piby Gas chromatog. 11.03 6.14 11.29 6.13 9.94 11.00 11.98 10.40 9.58 10.38 10.73 9.57 11.25

G CI AO AC 97.0 100.7 99.5 100.3 101.0 96.8 101.4 94.7 96.0 98.6 94.5 97.4 95.3

T a b l e II presents some data using the gas chromatographic p r o cedure and comparing the results to the standard A O A C procedure. T h e samples of p y r e t h r u m concentrate and A O A C analyses were supplied b y several of the principal p y r e t h r u m producers f r o m the U n i t e d States and abroad.



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N A T U R A L PEST CONTROL A G E N T S

Figure 10. Infrared spectrum of long-chain ester recovered from gas chromatography after hydrolysis and methylation of pyrethrum concentrate T h e columns labeled P I reflect the total of p y r e t h r i n I and cinerin I just as i n the A O A C procedure. T h e gas chromatographic results are i n terms of the total amount of the mixture b u t were analyzed as the m e t h y l ester of chrysanthemic acid. T h e present state of the determination of P H ( p y r e t h r i n II plus cinerin II) is not complete because of the erratic extractability of the dicarboxylic acids f r o m the hydrolysis mixture. T h e gas chromatographic pattern is distinct and straightforward. A s the extraction procedure f o r P I I is i m p r o v e d , the gas chromatographic method w i l l be more applicable. T h e present re c o v e r y of P I I is i n the range of 80 to 9 0 % . T h e average of the values shown i n T a b l e II f o r P I is 98.0%. It was recently f o u n d that the free monocarboxylic and dicar b o x y l i c acids derived f r o m p y r e t h r u m hydrolysis w i l l pass through the gas chromatograph and present suitable responses. W h e n this is f o l l o w e d u p , it is expected that the method w i l l be simplified and provided better over-all recoveries. Discussion Figure 1 presents the gas chromatographic responses obtained f r o m a p y r e t h r u m concentrate. Peak I has been identified as cinerin I, peak II as a cinerin-type c o m p o u n d , peak III as p y r e t h r i n I, peak


6.

BECKMAN AND ALLEN


63


I V as a cinerin-type c o m p o u n d , peak V as cinerin II, and peak V I as p y r e t h r i n II. It is evident that peak I V also contains another c o m ponent. T h e peaks beyond peak V I are k n o w n to be of the p y r e t h r i n type as they respond to the color test. T h i s gas chromatography pattern was obtained w i t h a 6-foot i4~inch c o l u m n packed w i t h 2 0 % S E - 3 0 o n 40-60-mesh Chromosorb P . Later w o r k p r o v i d e d g o o d resolution of the peaks w i t h a 2-foot V i - i n c h c o l u m n packed w i t h 2 0 % D o w - 1 1 silicone o i l o n 45-60-mesh Chromosorb P . Figures 2 through 9 are infrared spectra of fractions collected f r o m partition columns, gas chromatography, thin-layer chromatog raphy, or a combination of these separation techniques. Figure 10 is the infrared spectrum of a c o m p o u n d isolated b y gas chromatog raphy after hydrolysis of a p y r e t h r u m concentrate. In this case the c o m p o u n d is a long-chain ester. A l l the infrared spectra were made w i t h a P e r k i n - E l m e r M o d e l 221 instrument. T h e f o l l o w i n g operating parameters were used. A liquid demountable cell w i t h a 0.01-mm path length was employed. Resolution Response Gain Speed Suppression Scnle Prism

927 11.00 4.2 16 10 IX NaCl

T h e identity of certain of the compounds was confirmed b y available infrared data ( 7 , 2 2 ) . Literature Cited (1) Allen, P . T . , Beckman, H. F., Fudge, J. F., J. Agr. Food Chem. 10, 248 (1962). (2) Beckman, H. F., Anal. Chem. 26, 922 (1954). (3) Beckman, H. F., Allen, P . T., Berkenkotter, P., J. Gas Chromatοg. 1, 21 (August 1963). (4) B r o w n , N. C . , Hollinshead, D . T . , Phipers, R . F., W o o d , M. C . , Pyrethrum Post 4 (2), 13 (1957). (5) Chang, S. C., J. Agr. Food Chem. 9, 390 (1961). (6) Donegan, L . , G o d i n , P. J., Thain, Ε . M., Chem. Ind. (London) 1962, 1420. (7) Elliott, M., J. Appl. Chem. 11, 19 (1961). (8) Elliott, M., Olejniczak, J . S., Garner, J . J., Pyrethrum Post 6, 8 (1959). (9) G o d i n , P. J., Sleeman, R . M., Snary, M., Thain, Ε . M., Chem. Ind. (London) 1964, 371.


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(10) Harris, T. H., J. Assoc. Offic. Agr. Chemist 32, 684 (1949). (11) Kelsey, D . , Ibid., 43, 354 (1960). (12) Mitchell, W., Byrne, J. H. N., Tresadern, F . H., Analyst 88, 538 (1963). (13) Samuel, B. L., J. Assoc. Offic. Agr. Chemists. 35, 391 (1952). (14) Stahl, E . , Arch. Pharm. 293/65, 531 (1960). May

13,

1965.


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