792 Analyst, November, 1973, Vol. 98, pp. 792-796 A Spectrophotometric Method for the Micro- determination of Piperonyl Butoxide in the Presence of Pyrethrins BY H. M. BHAVNAGARY AND S. M. AHMED (Central Food Technological Research Institute, Mysore-zA , India) A rapid spectroyhotometric method has been developed for the micro- determination of piperonyl butoxide in the presence of a wide range of pyrethrins. A solution of the mixture of piperonyl butoxide and pyrethrins in a low-boiling fraction of light petroleum is evaporated on a water-bath until only a small portion of the solvent remains. The final traces of solvent are removed a t 50 "C and, to the residue containing piperonyl butoxide and pyrethrins, 18 per cent. m/V nitric acid is added in order to convert the piperonyl butoxide content into a soluble yellow-coloured compound.The quantitative colour reaction has a maximum absorption at 370nm, and the nzethod is applicable to the residues in the range 4 to 40 pg ml-1 of piperonyl butoxide. The method is also applicable to the residues of formu- lated products extracted from grains and paper coatings. PIPERONYL but oxide, 5- [2- (2-but oxyethoxy) et hoxyme t hyl] -6-propyl-l,3-benzodioxole,~ has been widely used in combination with pyrethrins as synergist in various formulations for the protection of grains and foodstuffs, in the control of household insect pests, in food processing plants and warehouses, and for fish preservation. The use of piperonyl butoxide has increased in recent years not only because of its synergism2 with pyrethrins but also because of the low toxicity of such combinations to warm-blooded animals3 A survey of the literature revealed that the gas-chromatographic methods4s5 for the quantitative deter- mination of piperonyl butoxide involved preliminary clean-up procedures, Jones, Ackermann and Webstere have reported a colorimetric method for the determination of piperonyl butoxide alone in which they heated it in purified kerosene with a solution of tannic acid in a mixture of phosphoric and glacial acetic acids in order to produce a quantitative blue colour reaction.This method could not be used in the presence of pyrethrins owing to interference by the latter. In an attempt to avoid this interference, they saponified the pyrethrins content of the mixtures before colour development, but this treatment reduced the colour value.They then suggested that piperonyl butoxide could be separated from pyrethrins by using the partition-chromatographic method. The interference of pyrethrins in the above method was also observed by Williams, Dale and Sweeney,' who considered that it was caused by the red colour produced in the reaction that occurred between the phosphoric acid and pyrethrins. Further, the method of Jones et al. was used by other worker^*^^ only after they had isolated the piperonyl butoxide by means of a column-chromatographic separation in order to prevent interference by the pyrethrins. This procedure again involved the use of additional steps, thus making the method more time consuming and increasing the error.A need was therefore felt to develop a simple spectrophotometric method for the deter- mination of piperonyl butoxide in the presence of pyrethrins. A quantitative colour reaction of nitric acid with piperonyl butoxide has accordingly been developed that forms the basis of a method for its determination. The method is highly sensitive and the coloured compound formed obeyed Beer's law for a suitable range of micro- amounts of piperonyl butoxide down to a minimum concentration of 4 pg ml-l. The method is free from intcrfrrence by pyrethrins. METHOD LIPPARITUS- S~ectro~hotometer-Beckman DU model, with a 10-mm cell. @ SAC a n d the authors.BHAVNAGARY AND AHMED 793 Colorimeter-Bausch and Lomb Spectronic 20, with 12 x 100-mm tubes. For con- venience, measurements were made at 375 nm rather than at the wavelength of maximum absorption (370 nm).Test-tubes-Ground-glass, stoppered test-tubes, with the dimensions 18 x 150 mm, were used for the colour reaction. REAGENTS- piperonyl butoxide. sufficient glass-distilled water to give 1 litre of solution. pyrethrins. compounds, as supplied by Reechem (RC, India). Piperonyl butoxide-Technical material with a minimum content of 80 per cent. of Nitric acid reagent-Add 250 ml of analytical-reagent grade nitric acid (sp. gr. 1.42) to Pyrethrum extract-A standardised commercial extract containing 20 per cent. in/m of Light petrolezcm-Extra pure, with boiling range 40 to 60 "C, and free from aromatic PREPARATION OF STANDARD SOLUTION- For the stock solution, dissolve 0-25 g of technical piperonyl butoxide in light petroleum and make the solution up to 250 ml in a standard flask.With a pipette, transfer 10 ml of the solution into a 100-ml standard flask and make the volume up to the mark with light petroleum, thus giving a solution containing 100 pg ml-l of piperonyl butoxide. PROCEDURE- Preparation of calibration gvaph-Transfer 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8 and %Om1 of the standard solution into glass-stoppered test-tubes and heat them on a water- bath until the residue obtained is solvent free. Add exactly 5 ml of the nitric acid reagent by means of a pipette and shake the mixture for 1 minute at a temperature not higher than 35 "C. Then measure the absorbance of the solution against a blank solution in the 12 x 100-mm tubes of the Bausch and Lomb colorimeter at 375 nm.Prepare a calibration graph from the results obtained. Preparation of test samples-Dissolve an accurately weighed amount of the samples con- taining pyrethrins and piperonyl butoxide in various proportions, such as 1 to 3, 1 to 5, 1 to 10 and 1 to 15, in light petroleum so as to give solutions that have piperonyl butoxide contents in the range 5 to 50 pg ml-l. Also, dissolve in the same solvent known amounts, within the above range, of piperonyl butoxide alone. COLORIMETRIC DETERMINATION- Transfer an aliquot of 5 ml of the test samples into 18 x 150-mm glass-stoppered test- tubes. Evaporate the solution on a water-bath until a small portion of the solvent remains, then continue to evaporate it in a water-bath adjusted to 50 "C until all of the solvent has been removed.To the residue add exactly 5 ml of the colour-forming reagent, stopper the test-tube and shake it well for 1 minute. Treat 5 ml each of the known sample and reagent blank in a similar manner. Then transfer the coloured solutions to the Bausch and Lomb colorimeter tubes and read the absorbance at 375 nm. Make all determinations in duplicate. CALCULATION OF RESULTS- The piperonyl butoxide content of the sample is calculated as follows. w (c - a) ( b - 4 Piperonyl butoxide content = where IVpg is the amount of piperonyl butoxide in the known sample, a the absorbance of the blank, b the absorbance of the known sample and c the ab; )r'itnce of t'is te;t solution. DISCUSSION COLOUR REAGENT- Under normal conditions, phenolic ethers undergo sulphonation, nitration and chlorina- These reactions were carried tionlo with electropliilic reagents by substitution in the ring.794 BHAVNAGARY AND AHMED : SPECTROPHOTOMETRIC MICRO-DETERMINATION [A%?aZySt, VOl.98 out on piperonyl butoxide by using the appropriate concentrated acids. It was observed that the reaction with nitric acid was quantitative and gave a yellow-coloured nitro compound that was soluble in the excess of nitric acid. Further investigations -with dilute nitric acid revealed that the reaction was complete when nitric acid in the concentration range 10 to 18 per cent. m/V was used for the nitration in aqueous medium. CHARACTERISTICS OF THE COLOUR REACTION- The absorption spectrum of the yellow-coloured nitrated piperonyl butoxide was deter- mined on the Beckman DU spectrophotometer in the wavelength range from 340 to 4-40 nm.Fig. 1 shows the spectra obtained with 30 and 60 pg of piperonyl butoxide present in 1 ml of solution, the maximum absorption occurring at wavelength 370 nm. The relationship between the concentration and the colour intensity obeyed Beer's law in the range 4 to 40 pg ml-l, the graph obtained being a straight line that passed through the origin. For the preparation of the standard graph, both technical and purified grades of piperonyl butoxide can be used as there was no quantitative difference between the colour reactions. The samples of piperonyl butoxide used should be free from organic solvents such as light petroleum, trichloroethylene and benzene, as the nitro compound partitions between these solvents and nitric acid.0.50 - 0.40 - a, C $ 0.30 - 54 Q 0.20 - 0.10.- 340 350 360 370 380 390 400 41 0 420 430 440 Wavelength/nm Fig. 1. Absorption spectra of piperonyl butoxide - nitric acid compound with reagent blank containing 14 per cent. of nitric acid: A, 60 pg ml-1 of piperonyl butoxide; and B, 30 pg ml-1 of piperonyl butoxide STABILITY OF THE COLOURED COMPOUND IN THE TEMPERATURE RANGE 25 TO 30 "C- The stability of the coloured product developed with 20 and 37 pg of piperonyl butoxide per millilitre of nitric acid solution was studied by reading the absorbances at 375nm at intervals of 1 minute for a period of 10 minutes and then at intervals of half an hour for a period of 10 hours. It was found to be stable for a period of up to 10 hours, beyond which time there was a gradual deterioration, as shown in Table I.EFFECT OF HEAT ON THE COLOUR REACTION- The absorbances of the colour developed at various temperatures between 20 and 100 "C with 165 pg of piperonyl butoxide, by adding 5 ml of the reagent, were studied. The relation- ship between the temperature of reaction and the absorbance at 375 nm is shown in Fig. 2. The coloured compound was stable up to a temperature of 35 O C , above which it decomposed 3teadily with increase in temperature. It is therefore necessary to maintain the temperature of the reaction mixture below 35 "C for satisfactory colour development.November, 19731 OF PIPERONYL BUTOXIDE I N THE PRESENCE OF PYRETHRINS TABLE I STABILITY OF THE COLOURED COMPOUND I N THE TEMPERATURE RANGE 25 TO 30 "c Absorbance* Time after development 1 minute 5 minutes 10 minutes 30 minutes 1 hour 5 hours 20 Gg ml-1 37 pg A1-1 of piperonyl of piperonyl butoxide butoxide 0.12 0.22 0.12 0.22 0.12 0.22 0.12 0.22 0.12 0.22 0.12 0.22 795 Time after ( development 10 hours 12 hours 15 hours 18 hours 2 1 hours 24 hours .Absorbance* - 20 pg ml-l 37 pg ml-1 3f piperonyl of piperonyl butoxide bu toxide 0.12 0.22 0.1 1 0.2 1 0.10 0.19 0.09 0-17 0.08 0.15 0.07 0.14 * Measured a t 375 nm in 12-mm tubes against water as reference. EFFECT OF THE NITRIC ACID REAGENT CONCENTRATION- To test-tubes each containing 150pg of piperonyl butoxide, 5 ml of nitric acid a t different concentrations were added and the colour was developed by shaking the mixture for 1 minute.In Fig. 3, the relationship between the concentration of nitric acid and the absorbance is shown. With dilute acids the colour reaction with piperonyl butoxide was incomplete. At an acid concentration of 10 per cent. m/V the absorbance reached a maximum and thereafter remained constant in the range 10 to 18 per cent. m/V, indicating that the colour reaction was complete. The concentration of the nitric acid used as the colour-forming reagent should therefore be above 10 per cent. m/V. 0.20 0.15 b 0.15 m W c m 2 0.10 % a Q 0.05 0 5 10 15 20 Ternperature/OC Nitric acid concentration, per cent. mlV Fig. 2. Effect of temperature on the Fig. 3. Effect of Concentration of absorbance of the piperonyl butoxide - nitric acid on the absorbance of nitric acid coloured compound the coloured compound containing 30 pg ml-l of piperonyl butoxide APPLICATION OF THE METHOD- The rapid spectrophotometric procedure developed has been applied satisfactorily to formulations containing pyrethrins and piperonyl butoxide in various proportions, It can also be used for the determination after extracting the combinations of pyrethrins and piperonyl butoxide from both sorptive and non-sorptive surfaces, such as glass, grains, paper coatings, etc.Either light petroleum or hexane can be used for the extraction and the solvent-free residues taken for the determination by the procedure described. CONCLUSIONS A sensitive and precise method for the determination of piperonyl butoxide in the It is based on the presence of pyrethrins by a spectrophotometric procedure is presented.796 BHAVNAGARY AND AHMED measurement of the yellow colour produced when the samples containing piperonyl butoxide are treated with 10 to 18 per cent.m/V nitric acid at a temperature not higher than 35 “C. There is no interference at any concentration level from pyrethrins, with which it is commonly used in formulations. The authors express their sincere thanks to Shri S. K. Majumder, Chairman, Discipline of Infestation Control and Pesticides, and Dr. H. A. B. Parpia, Director, Central Food Technological Research Institute, Mysore (India), for providing the necessary facilities during the investigations. The colour is stable and appears to be fairly specific for piperonyl butoxide. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. REFERENCES Wachs, H., Science, N.Y., 1947, 105, 530. Dove, W. E., Amer. J. Trofl. Med., 1947, 27, 339. Sarles, M. P., Dove, W. E., and Moore, D. M., Ibid., 1949, 29, 151. Miller, W. K., and Tweet, O., J . Agric. Fd Chern., 1967, 25, 931. Bevenue, A., and Kawano, Y . , J. Chromat., 1970, 50, 49. Jones, H. A., Ackermann, H. J., and Webster, M. E., J . Ass. Off. Agric. Chem., 1952, 35, 771. Williams, H. L., Dale, W. E., and Sweeney, J . P., Ibid., 1956, 39, 872. Williams, H. L., and Sweeney, J, P., Ibid., 1956, 39, 975. Secreast, M. F., and Cail, R. S., J . Agric. Fd Chem., 1971, 19, 192. Kirk, R. E., and Othmer, D. F., “Encyclopedia of Chemical Technology,” Volume 15, Interscience Received March 27th, 1972 Amended May 16th, 1973 Accepted June 19th, 1973 Publishers, New York, 1968, p. 168.