A?zalyst, June, 1967, Vol. 92, fifi. 375-381 375 Spectrophotometric Determination of Diquat and Paraquat in Aqueous Herbicide Formulations BY S. H. YUEN, J. E. BAGNESS* AND D. MYLEST (Im9erial Chemical Industvies Limited, Agricultural Division, Jealott’s H i l l Research Station, Bracknell, Berkshire) Methods are described for determining diquat and paraquat, singly and in admixture, in formulations. For determining diquat, ultraviolet absorptio- metry a t 310 mp in a sodium acetate buffer solution a t pH 4.05 is adopted. Paraquat is determined in a diluted solution by measuring the optical density, a t 600 mp, of the blue free radical produced by reduction with alkaline sodium dithionite. For analysing mixtures containing both diquat andparaquat, these methods are combined and use is made of a base-line correction procedure to compensate for interference of diquat in the determination of paraquat.The error for these methods is established to be within 3 2 per cent. DIQUAT and paraquat are the common names for l,l’-ethylene-2,2’-bipyridylium and 1,l’-di- methyl-4,4’-bipyridylium cations, respectively, which are manufactured in the form of diquat dibromide and paraquat dichloride or di(methy1 sulphate), and are the active components of commercial aqueous preparations known as Reglone: and Gramoxone. : A preparation incorporating both herbicides is known as Preeglone Extra. : The unique herbicidal properties of these compounds, their uses and advantages over conventional total herbicides have been described elsewhere.1 Methods have been reported for determining diquat and paraquat residues in water by ultraviolet absorptiometry,2 in food crops by spectropliotometry of the reduced ions394y5 and by polarography.6 Ultraviolet absorptiometry at 310 mp has been found to be specific and suitable for determining diquat in aqueous formulations, either alone or in the presence of paraquat.On reduction with alkaline sodium dithionite, both herbicides are converted to coloured free radicals that are relatively stable in an excess of reducing agent.’~* Solutions of the radical ions from diquat are green, the light absorption spectrum showing a sharp peak at 378 mp, tailing off through the visible region with an inflection from 410 to 440mp. Reduced paraquat is blue and the light absorption spectrum of the radical ions has a sharp peak at 394 mp, and a broad one at 600 mp.The blue colour of reduced paraquat is suffi- ciently stable in solution to enable paraquat to be determined differentially at 600mp. In formulations containing both diquat and paraquat, the latter can be determined if a base-line correction is applied to compensate for the absorption caused by the reduced diquat, which is virtually linear in the region from 520 to 700 mp. EXPERIMENTAL ULTRAVIOLET SPECTRA OF DIQIJAT AND PARAQUAT- Fig. 1 shows the ultraviolet spectra for diquat and paraquat in the buffer solution at pH 4.05, which contains 5.44 g of sodium acetate trihydrate and 9.5 ml of glacial acetic acid per litre, and is preferable to water in ensuring maximum stability and reproducibility of the peaks. At 310 mp, the maximum for diquat, the Ei& values for diquat and paraquat were found to be 1045-0 and 7-0, respectively, indicating that for a mixture containing equal cation weights of these herbicides, diquat should be determinable with as little as +0.7 per cent.error, caused by paraquat. Interference from a range of additives used in aqueous formula- tions, such as “wetters,” anti-foaming agents and corrosion inhibitors, has been found to be negligible at this wavelength. Consequently, ultraviolet absorptiometry at 310 mp has * Present address : Plant Protection Limited, Yalding, Kent. t Present address : Imperial Chemical Industries Limited, Dyestuffs Division, Blackley, Manchester. $ Registered trade marks of Plant Protection Limited.376 YUEN, BAGNESS AND MYLES : SPECTROPHOTOMETRIC DETERMINATION [Analyst, VOl.92 been used for determining diquat alone and in the presence of paraquat. The calibration graph over the range 0 to 0.6 mg was consistent and rectilinear, 0.6 mg of diquat in 100 ml of solution giving an optical density of 0.63 in a 1-cm optical cell. The absorption maximum of paraquat occurs at 257 mp, but this wavelength is unsuitable for determining paraquat, as in mixed formulations there is considerable overlap in this region from the absorption of diquat, and interference from certain additives, such as “wetters.” Wavelength, mp Fig. 1. Ultraviolet spectra of diquat and paraquat in sodium acetate buffer solution : curve A, 0-4mg of paraquat in 100ml; curve B, 0-4mg of diquat in 100 ml VISIBLE SPECTRA OF SOLUTIONS CONTAINING REDUCED DIQUAT AND PARAQUAT- The free radicals from diquat and paraquat, formed on reduction with alkaline sodium dithionite, are stable only in an excess of the reducing reagent.The colour may fade on standing owing to depletion of the dithionite in the immediate vicinity of the radical ions, which are then oxidised back to the parent cations. The maximum colour intensity for reduced paraquat can be restored immediately by gently swirling the solution. Vigorous shaking of the reduced solution causes rapid discharge of colour, owing to oxidation of the radical by atmospheric oxygen. On the other hand, if the sodium dithionite concentration is increased to above 2 per cent., over-reduction can occur with paraquat, leading to the formation of a less highly coloured dihydrobipyridyl derivative by uptake of 2 electrons.The stability of the colour produced on reduction is enhanced by decreasing the strength of the sodium hydroxide in the final solution. With solutions containing paraquat only, the optimum conditions for reduction have been found to require 1 per cent. sodium dithionite in 0.1 N sodium hydroxide (reagent I). However, work with formulations containing both diquat and paraquat has shown that rapid fading may occur unless a reagent consisting of 1 per cent. sodium dithionite in N sodium hydroxide (reagent 11) is used. Reagent I or I1 for appropriate analyses gives rise to paraquat radical colours that are stable for up to 3 hours from the time of mixing. The spectra produced by diquat and paraquat from 400 to 700mp are shown in Fig.2. In analysing solutions containing only paraquat, a calibration graph relating optical densities at 600 mp to concentrations has been found to be linear over a wide range. The colour is sufficiently stable to permit the use of differential absorptiometry. An optical density of 0.43 was obtained in a 1-cm optical cell with 1 mg of paraquat in 100 ml of reduced solution, measured against the 0-4-mg standard.June, 19671 OF DIQUAT AND PARAQUAT IN AQUEOUS HERBICIDE FORMULATIONS 377 Instead of ultraviolet absorptiometry, diquat, in the absence of paraquat, may be deter- mined absorptiometrically by reduction with 1 per cent. sodium dithionite in 2 N sodium hydroxide and measurement of the optical density at 430 mp.Wavelength, mp Fig. 2. Visible spectra of diquat and paraquat in reduced solution: curve A, 1 mg of paraquat in 100 ml; and curve B, 1 mg of diquat in 100 ml DETERMINATION OF PARAQUAT IN THE PRESENCE OF DIQUAT- As the spectrum of a reduced diquat solution is virtually linear from 520 to 700 mp, a base-line correction may be applied when paraquat is determined absorptiometrically at 600 mp, which involves the measurement of optical densities at three wavelength^.^ Wave- lengths at 550, 600 and 650 mp were selected, the shortest and longest lying at equal distances from the paraquat peak, 600 mp, and chosen so as to be as far apart as possible on either side of the maximum, consistent with the linear portion of the diquat spectrum. The corrected value of the observed optical density at 600 mp, designated EGOOM, is then calculated from the equation- EGOOM = E600 - 8 (E550 + E650) It should be noted that ESOOM is not the true, corrected optical density for paraquat, but a smaller value; this is immaterial as the calibration graph constructed from E600M is rectilinear and reproducible.A corrected optical density of 0.35, as compared with 0-88 when uncorrected, was given by a solution containing 1.2 mg of paraquat in 100ml of reduced solution, measured in a l-cm optical cell against the reagent blank. To assess the specificity of the base-line correction procedure for determining paraquat, mixtures containing 0.5 mg of paraquat and increasing amounts of diquat were reduced and measured at 550, 600 and 650 mp in a l-cm optical cell.Results found for E600M are shown in Table I, which indicates that for a mixture containing equal amounts of diquat and paraquat the error in the paraquat determination was insignificant, but a negative error would be expected when the diquat content greatly exceeded that of paraquat. TABLE I DETERMINATION OF EGOOM VALUES FOR MIXTURES CONTAINING DIQUAT AND PARAQUAT Paraquat added, mg per 100 ml 0-5 0.5 0.5 0.5 0.5 0.5 Diquat added, mg per 100 ml 0 0-05 0.1 0.5 2.0 5.0 Error, E,,,, found per cent. 0.154 - 0.154 0 0-154 0 0.155 0 0.148 - 3.9 0.141 - 8.4378 APPARATUS- YUEN, BAGNESS AND MYLES : SPECTROPHOTOMETRIC DETERMINATION [Analyst, Vol. 92 METHODS S$ectrophotometer-A Unicam SP500 is used. RE AGE XTS- Bufer solution, pH 4.05-Dissolve 5.44 g of sodium acetate trihydrate in water, add 9-5 ml of glacial acetic acid and dilute to 1 litre with water.Sodium dithionite, reagent I-Prepare a 1 per cent. w/v solution of sodium dithionite in 0.1 N sodium hydroxide. Sodium dithioizite, reagent 11-Prepare a 1 per cent. w/v solution of sodium dithionite in N sodium hydroxide. These reagents should not be kept for more than 3 hours. Sodium dithionite is unstable in the presence of moisture and should be stored in small bottles with tightly screwed lids in a desiccator. Take one bottle at a time for current use. Standard diquat solution-Prepare a stock solution by dissolving 0-1968 g of pure diquat dibromide monohydrate ( C12H12N2Br2.H20, molecular weight 362.1 ; 50-87 per cent. cation) in the buffer solution, diluting to 500 ml with buffer solution and mixing.Dilute 10.0ml of the stock solution to 100ml with buffer solution. 1 ml of solution = 0.02 mg of diquat. Standard paraquat solution-Dissolve 0.1097 g of pure paraquat di(methy1 sulphate) (C,,H2,N2S20,, molecular weight 408.4; 45.59 per cent. cation) or 0.0691 g of pure paraquat dichloride (C,2H14N2Cl,, molecular weight, 257.2 ; 72-40 per cent. cation) in water, dilute t o 500 ml with water and mix (paraquat salts are hygroscopic and should be dried at 100" C for 5 hours, then cooled in a desiccator before use). 1 ml of solution == 0.10 mg of paraquat. Both standard diquat and paraquat solutions should be prepared as required. PROCEDURE FOR ANALYSING FORMULATIONS CONTAINING PARAQUAT ONLY- Weigh accurately a portion of the well mixed sample containing about 1 g of paraquat into a 250-ml calibrated flask, dilute to the mark with water and mix.Call this solution A. Transfer 5.0 ml of this solution to a 200-ml calibrated flask, dilute to the mark with water and mix. Call this solution B. Transfer 10.0 ml of solution B, and 4.0, 6.0, 8-0 and 10.0 ml of standard paraquat solution, equivalent to 0-4, 0.6, 0.8 and 1.0 mg of paraquat, respectively, to five 100-ml calibrated flasks and dilute the content of each flask to about 80ml with water. Add to each flask, by a fast-running pipette, 10 ml of sodium dithionite, reagent I, dilute to the mark with water and mix by inverting the flask end-over-end three times. Mix each solution again in a similar way just before transferring it to the optical cell.Within 15 minutes of adding the reducing reagent, measure the optical densities of the solutions at 600mp in a 1-cm optical cell against the 0.4-mg standard as reference. Draw the calibration graph relating optical densities of standards to paraquat contents in milligrams, and read off the paraquat content of solution B; alternatively, compute the paraquat content by interpolation. Call this amount Xmg. 100 x x x s Weight of sample in grams paraquat content, per cent. w/v = where S is the specific gravity of the sample. PROCEDURE FOR ANALYSING FORMULATlONS CONTAINING DIQUAT ONLY OR hlIXTURES O F DIQUAT AND PARAQUAT- Determination of diquat-Transfer 10.0, 20.0 and 30.0 ml of standard diyuat solution, equivalent to 0.2, 0.4 and 0.6 mg of diquat, respectively, to three 100-ml calibrated flasks, dilute each to the mark with buffer solution and mix.Measure the optical densities of standards at 310 mp in a 1-cm silica cell against the buffer solution as reference, and draw the calibration graph relating optical densities to diquat contents in milligrams. Weigh accurately a portion of the well mixed sample containing about 0-5 g of diquat into a 250-ml calibrated flask, dilute to the mark with buffer solution and mix. Call thisJune, 196‘71 OF DIQUAT AND PARAQUAT IN AQUEOUS HERBICIDE FORMULATIONS 379 solution C. Transfer 10.0 ml of this solution to a 200-ml calibrated flask, dilute to the mark with buffer solution and mix. Call this solution D (solution D is also required for determining paraquat, if this is present). Transfer 5.0 ml of this solution to a 100-ml calibrated flask, dilute to the mark with buffer solution and mix.Measure the optical density of solution E at 310 mp in a l-cni silica cell, against the buffer solution as reference, and read off from the prepared calibration graph the diquat content of solution E ; alternatively, compute the diquat content by interpolation. Call this amount Y mg. Call this solution E. 100 x Y x s Weight of sample in grams Diquat content, per cent. w/v = where S is the specific gravity of the sample. Determination of paraqzmt-Transfer 4.0, 8.0 and 12.0 ml of standard paraquat solution, equivalent to 0-4, 0.8 and 1.2 mg of paraquat, respectively, to three 100-ml calibrated flasks, and add water to each flask, and to a fourth flask, to about 80ml.Add in turn to each flask 10 ml of sodium dithionite, reagent 11, from a fast-running pipette, dilute to the mark with water and mix by inverting the flask end-over-end three times. Transfer the solution to a l-cm optical cell and measure the optical densities at 550, 600 and 650 mp against the reagent blank as reference. Call these optical densities E550, E,oo and E650, respectively. Calculate the corrected optical densities, EGO, at BOO mp by the equation- Draw the calibration graph relating EGOOM to paraquat contents in milligrams. Transfer 10-0 ml of solution D to a 100-ml calibrated flask and dilute to about 80 ml with water. Add 10 ml of sodium dithionite, reagent 11, dilute to the mark with water and measure the optical densities at 550, 600 and 650mp, as described above.Calculate the corrected optical density, E600M, and read off from the prepared cali- bration graph the paraquat content in 10 ml of solution D; alternatively, compute the paraquat content by interpolation. E600hf = E600 - 4 (E550 + E650) Call this amount 2 mg. - 5 0 x z x s Weight of sample in grams Paraquat content, per cent. w/v = where S is the specific gravity of the sample. RESULTS AND DISCUSSION The accuracy of the recommended method was established by carrying out recovery experiments on laboratory made, formulated samples, consisting of appropriate corrosion inhibitors, “wetters” and anti-foaming agents, namely, 8 samples, each containing 20.0 per cent. w/v of diquat ; 16 samples, each containing 20.0 per cent.w/v of paraquat ; and 6 samples containing varying amounts of diquat and paraquat. These samples were analysed by the recommended methods (except the diquat formulations that were also assayed by differential absorptiometry, involving reduction of the herbicide with 1 per cent. sodium dithionite in 2 N sodium hydroxide and measurements of optical density at 430 mp in a 2-cm optical cell against the 0-5-mg standard, the calibration graph ranging from 0.5 to 2 mg of diquat in 100 ml of solution). Results obtained are shown in Tables 11, I11 and IV. TABLE I1 DETERMINATION OF DIQUAT IN LABORATORY MADE, FORMULATED SAMPLES Formulation Diquat dibromide . . Diquat dichloride . Diquat added, per cent. w/v 20.0 20-0 20.0 20.0 20-0 20.0 20.0 20.0 Diquat found, per cent.w/v Ultraviolet method 20-1 20.4 19.8 20.2 20.2 19.5 20-3 19-8 Dithionite method 20.0 20.2 19.6 10.8 19.7 19.8 19.9 20.3380 YUEN, BAGNESS AND MYLES : SPECTROPHOTOMETRIC DETERMINATION TABLE I11 DETERMINATION OF PARAQUAT IN LABORATORY MADE, FORMULATED SAMPLES [Analyst, Vol. 92 Paraquat added, Paraquat found, Formulation per cent. w/v per cent, w/v Paraquat di(methy1 sulphate) Without “wetter” .. 20.0 19.9 20.0 19.7 20.0 20.1 19.1 19.9 20.6 With “wetter” . . .. Paraquat dichloride Without “wetter” ,. With “wetter” . . .. 20.0 20.0 20.0 19-4 19.8 20.0 20.4 19-5 19.8 20.3 19.6 Table I1 shows that the percentage recoveries for diquat by ultraviolet absorptiometry ranged from 97-5 to 102.0, with a mean of 100.2 (standard deviation, k l . 5 per cent.).The dithionite method gave 98-5 to 101.5 per cent. recoveries, with a mean of 99.6 per cent. (standard deviation, k1.2 per cent.). The results obtained by these two methods are practically identical. Table I11 shows that the percentage recoveries for paraquat ranged from 95.5 to 103-0, with a mean of 99.4 (standard deviation, k l . 9 per cent.). With suitable adjustment of the dilution factors, these methods have been satisfactorily applied to a range of formulations, including water-soluble granules, and to technical liquors, and the precision has been found to be adequate for this type of analysis. TABLE IV DETERMINATION OF DIQUAT AND PARAQUAT IN LABORATORY MADE, FORMULATED SAMPLES Sample Diquat added, No. per cent. w/v 1 9.00 2 9.00 3 4.50 4 12.0 5 7.98 6 10.0 Diquat found, per cent.w/v 9.00, 8.96, 9.00, 9.00 8.96, 9.00, 8-96, 9.05 4-80, 4.80, 4.84, 4.77 11.9, 11.8, 11.8, 11-9 8.13, 8.08, 8.08, 8-10 10.1, 10.1, 10.1, 10-2 Paraquat added, per cent. w/v 9.00 8.99 8.99 2.95 9.99 7.97 Paraquat found, per cent. w/v 9.20, 8.80, 8.90, 9.00 9-05, 8.85, 8-90, 8.90 9-25, 9.05, 9-03, 9.23 2.80, 2.85, 2.88, 2.75 9.40, 9-45, 9.45, 9.30 8.01, 8.01, 8-05, 7.90 Table IV shows that for formulations containing equal amounts of diquat and paraquat recoveries were better than 98 per cent. for both herbicides. When the paraquat contents greatly exceeded those of diquat, or vice veysa, the results were less satisfactory. The recom- mended methods have been routinely applied to the commercial preparation, Preeglone Extra, which incorporates these herbicides at a 1 : 1 ratio, and agreement amongst replicate results usually lies within 0.3 per cent.of the mean. While the absorption band at 310nip in sodium acetate buffer solution may be taken as specific for diquat, the possibility of interference by substances other than paraquat must not be disregarded, particularly in technical liquors containing coloured impurities. Experience has, so far, shown such interference to be negligible. Likewise, no significant background absorption in the region of 310 mp has been observed in formulations as a result of the presence of a range of additives, including “wetters,” anti-foaming agents and corrosion inhibitors.June, 19671 OF DIQUAT AND PARAQUAT IN AQUEOUS HERBICIDE FORMULATIONS 381 Similar considerations apply to the procedure involving reduction by alkaline sodium dithionite, which appears to be highly specific for the bipyridylium ions. Any coloured impurities in aqueous formulations derived during manufacture from the technical diquat and paraquat concentrates have invariably been found to be diluted to negligible amounts before the final determination. We thank Dr. A. Calderbank for helpful criticism of the manuscript. 1. 2. 3. 4. 5. 6. 7. 8. 9. REFERENCES Springett, R. H., Outl. Agric., 1965, 4, 226. Faust, S. D., and Hunter, N. E., J . Amer. Wat. W k s Ass., 1965, 57, 1028. Calderbank, A., Morgan, C. B., and Yuen, S. H., Analyst, 1961, 86, 569. Calderbank, A., and Yuen, S. H., Ibid., 1966, 91, 625. -- , Ibid., 1965, 90, 99. Engilhardt, J., and McKinley, W. P., J . Agric. Fd Chew., 1966, 14, 377. Michaelis, L., and Hill, E. S., J . Gen. Physiol., 1931, 16, 859. Homer, R. F., and Tomlinson, T. E., Nature, 1959, 184, 2012. Allen, W. M., J . CEin. Endocr. Metab., 1950, 10, 71. Received January 4th, 1967