May, 19581 LINSTEAD SCIENCE AND POLITICS 283 The Spectrophotometric Determination of Parathion and p-Nitrophenol BY ELSA H JELT AND ANNA-LIISA RIUKULA (Institute of Forensic Medicine, University of Helsinki, Helsinki, Finland) Equal amounts of sample are heated in aqueous alkaline and in alkaline benzene solutions. In benzene solution, no hydrolysis of parathion occurs. The products are extracted with a (1 + 4) mixture of benzene and acetone, which dissolves both parathion and p-nitrophenol. The absorption of these solutions is measured over the wavelength range of 400 to 430 mp. In this region p-nitrophenol has a well defined absorption peak and parathion shows no absorption. The absorption of the unhydrolysed solution measures the background absorption from absorbing substances other than parathion.By subtracting this background absorption from that of the hydrolysed sample, the absorption of fi-nitrophenol formed by hydrolysis of parathion is measured. The ultra-violet spectrum of the sample in 94 per cent. ethanol is also measured, and $-nitrophenol is identified before and after hydrolysis by paper-ckromatographic methods. PARATHION (diethyl P-nitrophenyl phosphorothionate) is a commonly used insecticide. It is volatile in steam, sparingly soluble in water and soluble in most organic solvents, e g . , benzenef and light petroleum.2 When parathion is heated with alkali it is hydrolysed and the products include P-nitrophenol. The analytical methods for the identification and determination of parathion include paper-chromatographic method^^,^^^; methods based on the reactions of the aromatic nitro group6~7~* y 9 ; the spectrophotometric determination of parathion5sf0,1f s12,13; and, above all, methods based on the determination of p - n i t r ~ p h e n o l .~ ~ ~ ~ , ~ ~ ~ 918,19920284 H JELT AND MUKULA: THE SPECTROPHOTOMETRIC DETERMINATION [Vol. 83 1x1 some of the methods in which the identification and determination of parathion is based on the measurement of the absorption of parathion itself and that of p-nitrophenol after hydrolysis, the results are erroneous. For example, some commercial preparations of para- thion contain impurities, such as P-nitrophenol and emulsifying agents, which cause background absorption and thus affect the accuracy of the determinati0n.l ,11 These impurities must either be removed or compensation must be made for the background absorption caused by them.We have observed that the alkali salts of p-nitrophenol can be extracted from strong alkaline solutions with a mixture of benzene and acetone, and that the presence of benzene prevents hydrolysis of parathion. These facts, as well as the characteristic absorption in the ultra-violet region exhibited by parathion in ethanolic solution, and by P-nitrophenol in alkaline benzene - acetone solution in the visible region, make possible the determination of both P-nitrophenol and parathion, and also the measurement of the amount of background absorption in the visible region. The absorption spectra of e-nitrophenol and parathion are shown in Fig. 1. Wavelength, mp Fig.1. Absorption spectra: curve A, parathion in 94 per cent. ethanol; curve B, P-nitrophenol in 94 per cent. ethanol; curve C, P-nitrophenol in alkaline benzene - acetone solution It can be seen that both p-nitrophenol and parathion have absorption peaks in the ultra- violet region. In the visible region, p-nitrophenol has a well defined peak, whereas parathion shows no absorption. Beer’s law is obeyed over the concentration range used. The wave- length ranges used were from 235 to 320 mp and from 400 to 430 mp. In 94 per cent. ethanol the absorption peak of parathion occurs at 274mp, and that of P-nitrophenol at 315 mp. The absorption peak of P-nitrophenol in alkaline benzene - acetone solution varies between 408 and 422 mp, depending on the alkalinity of the solution.The principle of our method is as follows. The sample is extracted with an organic solvent, and three aliquots of equal size are each evaporated to dryness under reduced pressure. Benzene is added to another before the hydrolysis with alkali and both the reaction products are extracted with benzene - acetone mixture (1 + 4 by volume). The third residue is dissolved in 94 per cent. ethanol. The three solutions thus obtained are designated A , B and C. The optical densities of solutions A and B are measured at 420 mp against a blank solution identical with the test solution except that the sample has been omitted. The optical density of solution C is measured at 280mp against 94 per cent. ethanol. In the calculation, the following designations are used- A = the solution obtained after hydrolysis with alkali, B = the solution obtained after treatment with benzene and hydrolysis with alkali, C = the unhydrolysed ethanolic solution, One residue is hydrolysed with alkali.= parathion,May, 19583 OF PARATHION AND P-NITROPHENOL = P-nitrophenol, = extraneous absorbing materials, E = molar extinction coefficient, and c = molar concentration. 286 It is assumed that the initial sample contains parathion, p-nitrophenol and extraneous According to Beer's law, we have the following equations-- materials that also absorb in the same regions. At wavelength 420 mp, E, = AEP + EN + AEx . . . . . . * * (1) and E, = BEP + EN + BEz. . . . . . . . . ( 2 ) .. . . * - (3) At wavelength 280 mp, E, = EP + EN + Ex. (in which AEP = the optical-density contribution of parathion in solution A ) , ..A s no hydrolysis occurs when parathion is heated with alkali in the presence of benzene, and as parathion shows no absorption-at 420 mp, then- Ep = Ep = 0. Provided that the background absorption at 420 mp caused by extraneous materials amounts to the same value in both solution A and solution B, then- AEx = BEx* Subtraction of equation (2) from equation (1) gives- As 1 mole of P-nitrophenol is formed from 1 mole of parathion, then according to E, - E B = ,E~-+No. . . . . . . - - (4) Beer's law- cp = EA - (at wavelength 420 mp). . . .. * - (5) EN From equations (l), (2) and (ti), we can obtain the amount of p-nitrophenol formed on hydrolysis. This gives the true amount of P-nitrophenol, as the interfering absorbance due to extraneous materials is eliminated.On the other hand, if p-nitrophenol is the only substance present that absorbs at 420 mp, its concentration before hydrolysis is given by the equation-- . . .. * * (6) . . . . E B CN =- EN The concentrations of both parathion and p-nitrophenol are thus found and, as the molar extinction coefficients of both substances are known, the optical-density values at 280mp can be calculated. If the sum of these calculated values is equal to the optical density of the unhydrolysed solution C, then parathion and p-nitrophenol only are present in the sample, and their identification is confirmed both qualitatively and quantitatively. Even in the presence of extraneous absorbing materials, the amount of P-nitrophenol formed on hydrolysis can be reliably ascertained. EXPERIMEKTAL APPARATUS- All spectrophotometric measurements were made with a Beckman DU spectrophotometer with use of 1-cm silica and Corex cells.Hydrolysis of the parathion was effected in a special separating funnel constructed for this purpose and shown in Fig. 2. The extraction of the sample, the subsequent evaporation of the extract to dryness under reduced pressure, the hydrolysis in a closed system and the extraction of the hydrolysed product were performed in this vessel. REAGENTS- Parathion-This was further purified by dissolution in benzene and washing the benzene solution with dilute alkali, acid and water. The residue after evaporation of the benzene was dried in a vacuum-desiccator; it had dz!",C = 1.262,6 and n : O C = 1.5369.The molar extinction coefficient of parathion in 94 per cent. ethanol measured at 280 mp is 9426, which, for a concentration of 1 pg per ml, is 0.032.286 H JELT AND MUKULA: THE SPECTROPHOTOMETRIC DETERMINATION [Vol. 8 After recrystdlisation , the melting-poin was 112" C. The molar extinction coefficient in 94 per cent. ethanol measured at 280 m, is 4210, which, for a concentration of 1 ,ug per ml, is 0.030. p-Nitrophenol--The Merck product was used. Fig. 2. Reaction vessel for the hydrolysis All other reagents used were of recognised analytical grade. Pure parathion was kindly supplied by the Institut fur Gerichtliche Medizin an de of parathion Medizienischen Akademie Dusseldorf. PRELIMINARY EXPERIMENTS- Preliminary experiments for the spectrophotometric determination of p-nitrophenol i i alkaline benzene - acetone solution were performed, and the hydrolysis of parathion in alkalin solution was investigated both in the pres'ence and absence of benzene.The spectrophotometric detemination of p-nitrophenol in alkaline benzene - acetone solutio:, (1 + 4 by voZztwze)-To study the extraction of p-nitrophenol from a quantitative standpoint the following experiments were performed-- (i) $-Nitrophenol was dissolved in 5 or 10 rnl of alkaline benzene - acetone solution This was prepared by saturating the (1 + 4) mixture of benzene and acetone wit1 3.5 N potassium hydroxide. (ii) p-Nitrophenol was dissolved in 2ml of 3.5N potassium hydroxide and extractec from this solution with 10, 20 or three 10-ml portions of benzene - acetone mixture The solutions were diluted with alkaline benzene - acetone solution to a suitable volume clarified with acetone in the volumetric ratio 4 to 1, and the optical densities were measure( at 420mp.The results are shown in Table I. TABLE I EXTRACTION OF p-NITROPHENOL FROM POTASSIUM HYDROXIDE SOLUTION WITH BENZENE - ACETONE SOLUTION (1 + 4) Weight of p-nitrophenol in 3.6 N potassium hydroxide solution, pg 8 20 20 80 80 80 100 200 400 400 400 Concentration of p-nitro- phenol in final solution, Pg Per 0.64 1-60 3.20 6-40 3.20 1.28 8-00 6.40 6.40 6.40 6-40 Optical density a t 420 mp s&zz-= (4 (ii) - 0.106 0.265 - 0.536 - - 1.057 - 0-530 - 0.212 - 1.340 - 1.053" - 1*040* - 1.064 I 1.066 * The alkali lilyer w2s yellowish. Volume of benzene - acetone (1 + 4) used for extraction in experiment (ii), ml 10 - - 10 20 3 x 10 10 10 10 20 3 x 10 Extinction coefficient for 1 pg of p-nitrophenol per ml 0-166 0.166 0.168 0.165 0.166 0.166 0.168 0.165 0.163 0.166 0.167May, 19581 OF PARATHION AND P-NITROPHENOL 287 The results in Table I show that 100 pg of p-nitrophenol in 2 ml of 3.5 N potassium hydroxide are extracted quantitatively with 10 ml of benzene - acetone solution and amounts as high as 400 pg can be extracted quantitatively with 20 ml of benzene - acetone. Further, it can be seen that the absorption of P-nitrophenol in alkaline benzene - acetone solution obeys Beer's law over the concentration range of 0.64 to 8.0 pg per ml at 420 mp.Hydrolysis of ~a~athion-Ketelaar~~ states that the hydrolysis of parathion with alkali is a bimolecular reaction; the half-time at 15" C with N alkali is 32 minutes.Big$ reports that, when parathion is heated with ethanolic potassium hydroxide for 3 hours in sealed ampoules, the yield of p-nitrophenol is 87 per cent. In our work the hydrolysis was carried out by heating parathion with 3.5 N potassium hydroxide at 100" C in the reaction vessel shown in Fig. 2. To determine the percentage hydrolysis, different amounts of parathion were hydrolysed and the p-nitrophenol formed was determined as described previously. The results are given in Table 11. TABLE I1 HYDROLYSIS OF PARATHION IN 3.5 N POTASSIUM HYDROXIDE AT 100°C FOR 2 HOURS Amount of parathion present, tLg 40 41 60 80 82 82 120 123 160 Weight of p-nitrophenol found, CLg 18.2 18.0 27.4 35-2 36.3 37.5 52.8 55-2 71.4 Calculated amount of parathion, tLg 38.1 37.8 57.4 73.7 76.0 78.5 110.6 115.6 149.5 Hydrolysis, 95.3 94.9 95.6 92.1 92.7 95.7 92.2 94.0 93.4 % It can be seen that the hydrolysis varied between 92.1 and 95.7 per cent.The mean value, 93.7 per cent., is the same as that reported by Ketelaar. Hydrolysis of payathion in the presence of benxene-It was observed that benzene prevents the hydrolysis of parathion by alkali. Experiments were therefore made in which parathion was allowed to stand in benzene and was then boiled with strong alkali. In other experiments, parathion was allowed to stand in alkaline acetone solution both with and without the addition of benzene. Some of these benzene solutions were shaken with 2 ml of 3-5 N potassium hydroxide and the mixtures were kept at room temperature for 24 hours, with intermittent shaking.The other solutions were boiled under reflux with 2 ml of 3.5 N potassium hydroxide for 2 hours. The phases were separated and the benzene layer was washed with dilute acid and with water. One millilitre of the washed benzene solution was removed by pipette and evaporated to dryness under reduced pressure at 50" C. The residue after evaporation was dissolved in 94 per cent. ethanol and the optical density of the solution was measured at 280 mp. The results Different amounts of parathion in 2 ml of benzene were treated with alkali. are shown in Table 111.- TABLE I11 HYDROLYSIS OF PARATHION IN BENZENE WITH Amount recovered after 24 hours a t room of parathion, temperature, p g per 2 ml Initial concentration pg per 2 ml 80 79.1 120 118.6 160 159.7 2000 - 3.5 N POTASSIUM HYDROXIDE Amount recovered after 2 hours a t 100" C, pg per 2 ml 78.4 119-3 160.3 2001.1 From Table I11 it can be seen that no hydrolysis occurs when benzene is used as the In benzene, parathion can be heated with strong alkali, treated with dilute acid solvent.and with water, and freed from the organic solvent without any loss.288 H JELT AND MUKULA: THE SPECTROPHOTOMETRIC DETERMINATION [VOl. 83 In other experiments, different amounts of parathion were dissolved in 8 ml of acetone, and in 10 ml of the (1 + 4) benzene - acetone mixture. The solutions were shaken with 2ml of 3 6 N potassium hydroxide for 1 minute and set aside at room temperature for different lengths of time.The reaction times were 1, 3 and 24 hours. Two millilitres of benzene were then added to the solutions containing acetone as the solvent, and the solutions were shaken again. After separation of the phases, the benzene - acetone layer was prepared for spectrophotometric measurement as described under “Procedure.” The results are shown in Table IV. TABLE 1v HYDROLYSIS OF PARATHION IN ALKALINE SOLUTIONS OF ACETONE AND BENZENE: - ACETONE (1 + 4) Concentration Reaction of parathion, time, iug Per ml hours 1 40 1 4 1 80 With acetone as solvent- -- amount of Iptical density p-nitrophenol a t 420 mp formed, pg 0.006 0.0 0.022 1.7 0.117 8.8 0-016 0.0 0.047 3.5 0.22 1 16.7 With benzene - acetone solution (1 + 4) as solvent- amount of optical density fi-nitrophenol a t 420 mp formed, pg 0.002 0.0 0.007 0.0 0.051 3-63 0-004 0.0 0.007 0.0 0.109 8.2 A f \ The results in Table IV show that, in alkaline acetone solution, parathion is appreciably hydrolysed during the first 3 hours, and mobe than 40 per cent.is hydrolysed in 24 hours. In the presence of benzene, however, hydrolysis is retarded. No absorption due to p-nitrophenol can be detected after 3 hours and only about 20 per cent. of parathion is hydrolysed in 24 hours. The optical density of parathion together with the optical density due to extraneous materials can therefore be measured with0u.t interference from the hydrolysis, provided that the reaction time does not exceed 3 hours. METHOD PROCEDURE- Hydrolysis of pavathion-Parathion is dissolved in an organic solvent and an aliquot containing from 20 to 200 pg is transferred by pipette to the reaction vessel.The solution is evaporated to dryness under reduced pressure at 50” C. Two millilitres of 3.5 N potas- sium hydroxide are added to the residue, the stopper is fastened with a rubber band and the vessel is placed in a boiling-water bath. After 30 seconds, the stopcock is closed and heating is continued for 2 hours. After cooling, 5 to 20 ml (V,) of benzene - acetone solution (1 + 4) are added, the mixture is shaken for 1 minute and then set aside for 10 minutes, after which the alkali layer is removed, Four millilitres of the benzene - acetone layer are removed by pipette and mixed with 1 ml of pure acetone. This solution is used for the spectro- photometric determination.The optical density of this solution, which is designated solution A , remains unchanged for at least 24 hours. Determination of p-nitrophenol and the background absorption-An aliquot of equal size to that used for the hydrolysis is placed bsy pipette in the reaction vessel and evaporated to dryness. From 1 to 4 ml of benzene are added to the residue, and then 2 ml of 3.5 N potassium hydroxide, A reflux condenser is attached to the vessel and the solution is heated under reflux in a water bath for 2 hours. After it has cooled, the solution is shaken with 4 to 16ml of acetone, and the layers are separated. Four millilitres of the benzene- acetone layer are removed by pipette and. diluted with 1 ml of acetone.This solution is used for the spectrophotometric determination. The optical density of this solution, which is designated solution B, remains unchanged for 3 hours. PreParation of the blank solution-Ten millilitres of benzene - acetone solution (1 + 4) are shaken with 2 ml of 3.5 N potassium hydroxide. The layers are separated and 4 ml of the benzene - acetone layer are removed by pipette and diluted with 1 ml of acetone. This is the blank solution. Its optical density remains unchanged for at least 24 hours.May, 1958] OF PARATHION AND p-NITROPHENOL 289 Preparation of the control solution-An aliquot of equal size to that used for the hydrolysis is evaporated to dryness. The residue is dissolved in 5 to 20 ml (V,) of 94 per cent. ethanol. This is solution C.Measurement of optical density-The optical densities of solutions A and B are measured against the blank solution at 420 mp. This gives the values of E, and E, in equations (1) and (a), p. 285. The optical density of solution C is measured at 280 mp against 94 per cent. ethanol. This gives the value for E, in equation (3). Pa9er-chromatographic detection of p-nitrophenol-The hydrolysed and unhydrolysed solutions are chromatographed. By using the ascending-solvent technique with a (1 + 1) mixture of isobutyl and isoamyl alcohols saturated with ammonia as solvent; the RF value for p-nitrophenol is 0.48. The chromatograms are allowed to develop at 23" C for 18 hours. The spots are detected by spraying with ammonia or alkali. CALCULATIONS- To calculate the results, the constants for a concentration of 1 pg per ml are as follows- For P-nitrophenol in solutions A and B at 420 mp, eN = 0.166.For p-nitrophenol in ethanol at 280mp, E~ = 0.0303. For parathion in ethanol at 280 mp, ep = 0.0324. The percentage hydrolysis is 93.7, and the molecular weights of parathion and +nitro- Hence the concentration of parathion is given by- If V , is the volume of benzene - acetone solution, and V , the volume of ethanol used, phenol are 291.3 and 139.1, respectively. cp = 2-23 x concentration of p-nitrophenol in solution A . then, by applying equation (5), the amount of parathion per aliquot is given by- The corresponding optical density measured at 280 mp is- 16.8 x V l x ( E A - EB) pg. V Ep = 0.545 x -2 x (EA - E,). v2 From equation (6)- Weight of P-nitrophenol per aliquot = 7.54 x V , x E, pg.The corresponding optical density measured at 280 mp is given by- V If the sum of the calculated values for EP and E N is equal to Ec (all measured at 280 mp), If this sum is smaller E N = 0.227 x 2 x E,. v 2 the amounts of both parathion and p-nitrophenol are confirmed. than E,, the amount of P-nitrophenol formed on hydrolysis can be determined. RESULTS Prepared mixtures of parathion and fi-nitrophenol and some commercial preparations were analysed. The results of four such analyses calculated in micrograms per millilitre are given below. (i) A solution containing 80 pg of parathion and 21-9 pg of p-nitrophenol in 1 ml of benzene was prepared. The presence of fi-nitrophenol was detected, both before and after hydrolysis , by paper-chromatographic techniques.EA was 0-768 and EB was 0-286, therefore EA - E, was 0.482. The amount of +-nitro- phenol found was 21.6 pg, for which, at 280 mp, E N = 0.065. The amount of parathion found was 80.9 pg, for which, at 280 mp, EP = 0.263. It was found that, at 280 mp, Ec = 0-336. From these results it can be concluded that the sample contains 80.9pg of parathion and 21-6 pg of p-nitrophenol. (ii) A commercial powder contains about 20 per cent. of parathion, according to specification. An extract containing 200 pg of the sample in 1 ml of benzene was prepared. The paper-chromatographic study showed that, before hydrolysis, no fi-nitrophenol was present, but after hydrolysis a positive result was obtained. In each analysis both V , and V , were 10ml. Ep + E N z= 0.328.290 HJELT AND MUKULA [Vol.83 EA was 0.284 and E, was 0.001, therefore EA - E, was 0.283. No p-nitrophenol was found, and the amount of parathion found was 47.5 pg, for which, at 280 mp, EP = 0.154. It was found that, at 280 mp, E, = 0.15'7. From these results it can be concluded that the sample contains about 23.7 per cent. of parathion. (iii) A commercial fluid contains about 35 per cent. of parathion, according to speci- fication. An extract containing 133.9 pg of the sample in 1 ml of benzene was prepared. The paper-chromatographic study showed the presence of (P-nitrophenol both before and after hydrolysis. EA was 0.263 and E, was 0.036, therefore EA - EB was 0.227. The amount of p-nitro- phenol found was 2-7 pg, for which, at 280 mp, EN = 0.008, and the amount of parathion found was 38.2 pg, for which E, = 0.124. It was found that Ec = 0.161.From these results it can be concluded that the sample contains about 28.5 per cent. of parathion, not more than 2.0 per cent. of P-nitrophenol and extraneous absorbing materials. (iv) A commercial fluid contains 33.5 per cent. of parathion, according to specification. An extract containing 126-9 pg of the sample in 1 ml of benzene was prepared. The paper- chromatographic study showed the presence of P-nitrophenol both before and after hydrolysis. E, was 0.346 and E, was 0.092, therefore E, - EB was 0.254. The amount of $-nitro- phenol found was 6.9 pg, for which, at 280 mp, EN = 0.021. The amount of parathion found was 42-7 pg, for which E, = 0.138.It was found that Ec = 0.235. From these results it can be concluded that the sample contains about 33.6 per cent. of parathion, not more than 5.4 per cent. of (P-nitrophenol and extraneous absorbing materials. Ep + E N = 0.154 + 0 = 0.154. Ep + EN = 0.132. Ep + EN = 0.159. CONCLUSIONS By using the proposed method the p-nitrophenol formed on hydrolysis of parathion can be determined. The background absorption does not interfere with the determination, as it can be measured separately. The method is applicable to the determination of parathion in commercial preparations. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. REFERENCES Biggs, A. I., Analyst, 1965, 80, 279. Edwards, F. I., jun., and Hall, S. A., U.S. Patent 2,606,279 (July 29th, 1952); Chem. Abstr., Metcalf, R. L., and March, R. B., Science, 1953, 117, 527. Cook, J. W., J . Ass. 08. Agric. Chem., 1954, 37, 987. Fiori, A., Nature, 1956, 178, 423 Averell, P. R., and Norris, M. V., Anal. Chem., 1948, 20, 753. O'Keeffe, K., and Averell, P. R., Ibid., 1951, 23, 1167. Yamamura, J., and Niwase, T., Kagaku to, Sbsa [Science and Crime Detection], 1954, 7, 195; Chem. Paulus, W., Mallach, H. J., and Janitzki, U., Arzneimittel-Forsch., 1955, 5, 241. Sokol, F., Chem. Zvesti, 1953, 7 , 429. Derkosch, J., Jansch, H., Leutner, R., arid Mayer, F. X., Monatsh. Chem., 1954, 85, 684. Kotakemori, M., Ann. Re$. Takamine Lab., 1954, 6, 146; Chem. Abstr., 1955, 49, 16307~. Schmidt, G., Arch. ToxicoE., 1955, 15, 361. Eicken, S. V., Angew. Chem., 1954, 66, 551. Pfeil, E., and Goldbach, H. J., Klin. Woclzschr., 1953, 31, 1011. Jachimovicz, T., &err. Chem.-Ztg, 1954, 55, 190. Briegleb, G., and Angerer, G., Angew. Chem., 1952, 64, 685. Schroeder, W. A., Wilcox, P. E., Trueblood, K. N., and Dekker, A. 0.' Anal. Chem., 1951, 23, Ketelaar, J. A. A., Rec. Trav. Chim. PaysBas, 1950, 69, 649. Buckley, R., and Colthurst, J. P., Analyst, 1954, 79, 285. 1952,46, 10525~. Abstr., 1955, 49, 76301. 1746. Received May 31st, 1967