Identification of 3,4-Methylenedioxyphenyl Synergists by Reversed-Phase Paper Chromatography MORTON BEROZA Entomology Research Branch,
Agricultural Research Service,
Insecticide synergists or other w m p o u n d s c o n t a i n i o g the 3,4-methylenedioxypheaylgroupcan beidentified in the 2- to 10-7 range by c h m m a t o g r a p h y on paper strips impregnated with paraffin oil, with 30% acetic acid a s the developing solution. The position of the com. pound on the strip is d e t e r m i n e d b y direct 8pectro. photometry; the difference in absorbance a t two wave lengths, w r r e s p o n d i n g to a high a n d low absorbance value of the synergist, is used. This eliminates tluetu. ations i n absorbance due t o irregularities of the paper. b u t shows up the large absorbance differences of the synergists. The procedure also reduces interference f r o m o t h e r materials usually present in inseetioidr formulations.
F .
OLLOWING the discovery of the importance of the methyl. enedioxyphenyl group in contributing toward synergisrrI with pyrethrins (7), a number of excellent synergists containing this group (11, 18, 14) were produced commercially and are noa in widespread use. Several methods have been advanced for the determinatior of these methylenedioxyphenyl synergists (1-3). These pro. cedures do not distinguish between the various synergists, although a method for the determination of piperonyl butoxide that does not respond ta other commercial synergists has beer published (8). A means of identifying synergists or othei compounds containing the methylenedioxyphenyl group would not only extend the usefulness of the known procedures but would he useful for regulatory and far research purposes.
U. 5. Department of Agriculture, Beltrrille, Md.
zero absorbance over a suitable portion of the chromatogram were readily obtained without any special washing of the paper. This technique may have wide applicability for detecting or determining oompaunds by direct photometry af paper strips. APPARATUS
Spectrophotometer, Beckman Model DU. Adapter (Figure 1). This fits into the cell compartment of the spectrophotometer and is essentiallv the same in operation
~~~
~
~~
~~~~
ultra&hk measu;&ents, and t o give greeter ease and certainty to the operation. Except for the indicating spindle, D,which is stainless steel and roughened to prevent slippage, the unit is made oi brass. All the spindles ( A B C, D,E ) are inch in diameter. The bases of solibshaft &dles A and E are held a t the proper distance with aiittle salde;. Free rotation of A and E is prevented by serewing the knobs onto an instrument lock washer and locking the knob with a set screw. A scale in millimeters, 1 cm. on each side of the beam center line, is scribed on base plate F to facilitate checking the position of the beam periodically from lines marked a t the edge of the paper. Ii paper l
Chrom&graphic Chamber. A resin reaction apparatus (Corning Citaiog No. 6947, 4000-ml. capacity) is liGd with filter paper that dips into the solvent. Papers, suspended in the flask from the cover. are dinned inch into the solvent and developed by the asc6ndiug teihnique. Any similar setup should be equally satiafactory. MATERIALS
Impregnating Solution. This contains 3% purified p a r a 5 oil in petroleum ether (boiling point 60" to 70' C.). Synergists. Sulfoxide, piperonyl cyclonene, and n-propyl isome were technical products. The piperonyl butoxide wm a pure sample. Sesamin and sesamolin were pure compounds isolated from sesame oil. Solvents, c. P., redistilled. Filter paper, Whatman No. 1 rolls, 1- and ll/*-inch widths. PROCEDURE
Figure 1. Adapter for direct spectrophot o m e t r y of paper strips
A method was devised whereby the synergists, subjected to reversed-phase paper chromatography, were detected by direct spectronhotometry of the paper strips. An adapter similar to
wave lengths, corresponding to high and low absorbance values of the synergists. The absorbance difference was plotted against position on the paper. By this means it was possible to correct for the irregularities of the paper, and blanks having a near-
Draw the filter paper strips through the impregnating solution and hang to dry from the end first introduced. Spot solutions containing about 10 y of synergist l a / 4 inches from this end with a micropipet, Usually 1 pl. of a 1% solution in acetone is used and about I/s hour allowed for the acetone to evaporate. With paper 1 inch wide one solution, spotted on the center line, may he
paper with tGeezers or bv the edges after drving Develop the chromatkram &h 30% ace& acid until the solvent front ha8 moved ahout 15 cm. past the spotting paint or origin ( l Y 4 hours). Remove the paper, mark the solvent irant with B Dencil. and d r v in a hood for at least 1 hour. Mark the edge of the daper at"esch centimeter between orkin and front,
wave lendh'at 289 mp, andAahsorhancescale (density knob) a i 0.100 to 0.200; open the shutter, and balance by adjusting the slit width and sensitivity knobs. Record slit width. Do not.
1550
V O L U M E 28, NO. 10, O C T O B E R 1 9 5 6 adjust senstivity knob after this point. Without moving the paper or changing the absorbance (density dial), change the wave length to 305 mp and adjust slit width to balance the instrument. Record this slit width, too. The paper may now be advanced to the origin and the absorbance determined a t the two wave lengths with the corresponding slit widths for each position. (Setting of the wave length and slit width knobs may be speeded up by drilling a hole in the base of these knobs toward their center and forcibly inserting a 3-inch-long rod. At the proper settings crayon marks or mechanical stops are placed on the spectrophotometer.) Plot absorbance difference against position. Compare results vith known synergists, preferably run a t the same time. Solutions containing interfering materials, especially alkylated naphthalenes, should be spotted and the paper either left in a hood overnight or allowed to remain in an oven set a t 100" C. for 1 hour before developing. The data obtained with the six synergists are summarized in Figure 2.
1551
I '
I
I
I
I
I
1
DISCUSSION
The 3,4-methylenedioxyphenyl group is a stable structure. Colorimetric methods for its detection and estimation depend upon the hydrolytic cleavage of the methylenedioxyl group by strong acid to give formaldehyde, Tvhich is determined in the same acid medium (1-3). The high concentration of arid destroys the k$ VALUE paper, so that detection of the synergists by this means is not feasible. Figure 2. Paper chromatography of synergists (107)and blanks Because the ultraviolet spectra of the A . Absorbance of blank at 289 mp synergists (Figure 3) disclosed a high absorbB . Same as A but absorbance at 305 mp subtracted C . Piperonyl butoxide ance a t 289 mp, direct photometry offered a D . Sulfoxide means of detection. But the irregularity of E. n-Propyl isome F . Sesamin the paper, probably caused by varying thickG. Sesamolin H. Piperonyl cyclonene ness, caused large fluctuations in the absorbance obtained, even for blanks. Further examination of the spectra showed that the synergists have a low absorbance a t 305 mp. By setting the matograms of the unknown with the known, preferably on the slit widths for the 289- and 305-mp wave lengths to give same paper. the same absorbances a t a point where there is no synergist I n setting up the solvent system(s) to be used for the identifi-that is, below the origin-and determining the absorbance cation of the synergists on paper, it was recognized that the best differences of the paper a t the tn-o Tvave lengths with the synergists have an appreciable solubility in paraffin hydrocarbons corresponding slit Ridths, the effect of the paper is canceled that are widely used for diluting insecticide mixtures. A reout (compare blanks A and B , Figure 2), whereas the synerversed-phase system with paraffin oil as the immobile phase gists show large absorbance differences. Only in the region 2 to appeared most promising. Furthermore, the oil made the 3 cm. behind the front does some absorbance appear, but the paper less opaque to direct photometry. A preliminary investigasynergists do not reach this region with the 30% acetic acid tion was set up t o determine the ability of a series of polar dedeveloping solution. With developing solvents giving higher R, veloping solvents to resolve the synergists. The results are values, the interfering absorbance appears to be constant and given in Table I. therefore may be subtracted. Solvent systems which gave R, values that were too high or too This method of eliminating fluctuations in absorbance caused low or caused too much spreading of zones are not given-for by irregularity of the paper makes it possible to detect lesser instance, Tyater-saturated nitromethane gave R, values that were amounts of ultraviolet-absorbing materials. too high. Rf values could be regulated by the amount of water One of the difficulties in identifying the commercial synergists added to the polar phase. These preliminary runs were made in is due to the fact that they are mixtures rather than pure coman unlined I-liter glass-stoppered graduated cylinder except for pounds. The chromatograms in Figure 2 demonstrate this fact. the run with dimethyl formamide, which n-as made in the unThus, piperonyl butoxide, a pure sample in this study, gave a lined chromatographic chamber. It is recognized that R, chromatogram typical of a pure substance. Piperonyl cyclonene, values obtained in one chromatographic chamber may differ from those obtained in another. known to be a mixture, gave a rurve n i t h a broad peak. Sulfoxide, thought to be a single substance, apparently contains a t The best distinction between the six synergists was possible least t n o major constituents, and n-propyl isome contains even with 30% acetic acid, based on differences in Rfvalues and least more. With all the developing solutions tried poorest results spreading of zones. In case any doubt arises in distinguishing were obtained for n-propyl isome, which gave no well-defined between synergists, one of the other developing solutions listed in peaks unless the Rj value was near zero or 1.00. I n spite of their Table I may be used. For instance, with 40% acetic acid ninhomogeneity, the synergists give patterns that are readily propyl isome spreads with gentle maxima and is readily distinidentified, final confirmation being made by comparing the chroguished from piperonyl cyclonene, which gives one sharp peak.
ANALYTICAL CHEMISTRY
1552
I'
' 1 . '
'
'
'
'
'
'
'
"
"
1
Temperature was not controlled, although the rvperiments were usually conducted between 25 and 28" C. Quantitative determinations on paper are usually of limited accuracy. By summing the absorbance differences of zones (area under curve), synergists alone could be determined with a 5% accuracy, whereas similar determinations on synergists in formulations were unreliable. INTERFERENCES
To determine TThich of the materials generally formulated with synergists interferes, chromatograms containing 10 y of each of the commercial synergists were run in or with each of the following: 1 gl. of deodorized kerosine, 80 y of alkyl thiocyanates, 20 y of DDT, and 2 y of pyreth230 250 270 290 310 330 350 rins. The R, values differed a maximum of MI LLI M IC RO NS only 0.03 from those obtained on the synergists alone, and in some cases the curves tended to drag Figure 3. Ultraviolet absorption spectra of synergists (0.08 mg./ a bit. The alkyl thiocyanates gave a slight absorbml.) in 2,2,4-trimethylpentane ance difference near the origin. These effects did Values at 5-mp intervals not interfere with the identification of the synergist. A . Piperonyl cyclonene I n the presence of 60 y of alkylated naphthalenes B. Sulfoxide C. Piperonyl butoxide the decrease in Rr value and dragging effect were D. n-Propyl isome Spectra of sesamin and sesamolin in (6) pronounced. Allowing the spotted paper to remain in a hood overnight prior to development snfflciently minimizes this interference (probably by evaporation). .hother means of overcoming this interference is to An automatic scanning device for the Cary recording spectroallow the paper to remain in an oven a t 100" C. for 1hour before photometer has been used to determine total ultraviolet absorpdevelopment. The latter treatment changes the Rrvalue slightly, tion of vitamins against position on paper strips a t several wave so that a known should be run under identical conditions, preflengths ( 4 , 6 , 10). With this device strips are scanned before and erably on the same paper, for confirmation of results. after development to correct for the irregularity of the paper. Satisfactory identifications of the synergists in typical fly sprays-1% of synergist, 1% of pyrethrum extract ( 2 0 % ) , and 2% of D D T in deodorized kerosine-n-ere readily made. Good results were also obtained with the following lowTable I . Rr Values of Synergists Developed with Different pressure aerosol formations; 2y0 pyrethrum extract ( 2 0 % ) , 2% Aqueous Solutions DDT, 1% synergist, 5% aromatic petroleum derivative solvent, PiperPiper5% deodorized kerosine, and 85% propellant (Type I1 of Federal Solution onyl onyl (V./V.) ButoxSulCyclon-Propyl Sega- SesaSpecification 0-1-508 dated June 4, 1954). -4fter the propellant 70 ide foxide nene Isome min molin was evaporated, the residue was diluted with acetone so that tlceticacid 70 0.87 0.89 0.95 a 0.98 0.94 50 0.74 0.87 0.75 b 0.88 0.88 the synergist was present in about 1% concentration (u-./v ), 40 0 54 0.81 0.42 b 0.84 0.78 and 1 pl. was applied to the paper. 30 0.21 0.12 0.105b 0.64 0.47 C
illethanol Acetonitrile
70 50 35
0.64 0.20 0.25
0.86 0.62 0.61
0.62 0.14 0.22
Dimethylformamide
50
0.24
0.75
0.28
b
0.66 0.60 0.50d 0.3Od 0.66d 0.55
b
0.77
6
0.16b
0.76
ACKNOWLEDGMENT
The author gratefully acknowledges the assistance of Joseph
F. hlullins, Federal Extension Service, Beltsville, Md., in construction of the adapter. LITERATURE CITED
This method would be unsatisfactory in the present analyses. The procedure of determining the absorbance difference a t two Ivave lengths corresponding to high and low absorption of the compound lends a greater degree of specificity to the analysis and this was particularly evident when the insecticide formulations were analyzed. Thus, alkylated naphthalenes, even after heating 1 hour a t 100" C., contributed much absorbance near the origin, although the absorbance differences a t the two wave lengths were small. Of course, use of the automatic setup a t two different wave lengths would greatly speed up the present type of analysis. Identifications of a t least 2 y of synergist were readily made, little difference in R, value being noted in the 2- to 10-7 range. If the synergist is introduced in a nonvolatile solvent, no more than 1 pl. should be spotted, to avoid excessive spreading.
( I ) Beroaa, AI., A N A L . CHEX 26, 1970 (1954). (2) Beroea, IT.,J . -4gr. Food Chern. 4, 53 (1955). (3) Blum, AT. S., I b i d . , 3, 122 (1955). (4) Brown, J. A , , ~ A L CHEM. . 25, 771 (1953). (5) Brown, J. A . , Xarsh, M . AI., Ibid., 24, 1952 (1952). (6) Budowski, P., O'Connor, R. T., Field, E. T., J . A m . Oil Chemists' S O C28, . 51 (1951). (7) Haller, H. L., LaForge, F. B., Sullivan, TV. N., J . OTQ.Chem. 7, 185 (1942). (8) Jones, H. A , dckermann, H. J., Webster, 31. E., J . Assoc. O&. A g r . Chemists 35, 771 (1952). (9) Palladini. -4. C., Leloir, L. F., ASAL.CHEX 24, 1024 (1952). (IO) Parke. T. V., Davis, TV.W., Ibid., 2019 (1932). (11) Synerholm. A I . E., Hartzell, 4., Coiltribs. Bouce Thompsor~I n s t . 14, 79 (1945). (12) Synerholm, 11. E., Hartzell, A , Cullmaim, V., I b i d . , 15, 35 (1947). (13) Tennent, D. M., Whitla, J. B., Florey, K., ; ~ X A L . CHEM.23, 1748 (1951). (14) Waohs, H . , Science 105, 530 (1947).
RECEIVED for review February
24, 1956.
Accepted June 12, 1956.
