1557 Analyst, July 1994, Vol. 11 9

Kinetic-Spectrophotometric Determination of Diquat Based on a Charge-transfer Complex With Cysteine

Tomas Pbrez-Ruiz, Carmen Martinez-Lozano, Virginia Tom& and Rocio Casaj6s Department of Analytical Chemistry, Faculty of Chemistry, University of Murcia, 30071 Murcia, Spain

The reaction involving the formation of a charge-transfer complex between diquat and cysteine was studied. The initial rate of the reaction, measured as a change in absorbance at 495 nm during 60 s, was linearly related to diquat concentrations from 0.5 to 28.0 pg cm-3. The applicability of the method was demonstrated by the determination of diquat in commercial herbicides, waters, soils and potatoes.

Keywords: Diquat; kinetic method; cysteine

Introduction

Diquat (1,l '-ethylene 2,2'-dipyridylium dibromide) is a non- selective fast-acting herbicide, which has found extensive industrial and domestic applications. Its herbicidal activity appears to depend, in part, on the ease of one-electron reduction in photosynthesizing plant tissue.' It is also used as a desiccant to aid harvesting and to assist in the conservation of forage. This herbicide is extremely toxic to man.*

The determination of diquat in herbicide formulations,3 water,4 urine,5.6 blood5 and agricultural products7-9 has been reported. The methods used include ~pectrophotometry,7.8~~0 spectrofluorimetryll312 gas chromatography,13 liquid chroma- tography14 and polarography.l5,16 Other methods include direct potentiometry with ion-selective electrodes,l7J8 elec- tron spin resonance,l9 flow injection12720 and photo-kinetic met hods .21

Spectrophotometric methods are the most often used. They normally involve utlraviolet absorptiometry at 310 nmlO or visible spectrophotometry of the green radical (E = 4.1 X 103 dm3 mol-1 cm-1 at 430 nm) obtained by reduction with alkaline sodium dithionite7 or hydroxostannate(ii) .22 This radical is stable only in an excess of the reducing agent.

Diquat yields a fairly stable red complex with 2-mercapto- ethanol, dithiothreitol or cysteine.23 This complex (E = 2 X 104 dm3 mol-l cm-1 at 495 nm) has been considered as a reduced form of diquat, and it was found necessary to wait at least 10-12 min after mixing the reactants for its complete formation to take place. The absorption at 495 nm has been used for the determination of this herbicide.23

All viologens (among them paraquat and diquat) must be fairly good electron acceptors because of the stability of their free-radical ions24 and they have a strong tendency to form charge-transfer complexes with donor anions.25 Hence, para- quat forms charge-transfer complexes with a wide range of anions [halides, sulfides, tetraphenylborate, benzilate, hexacyanoferrate(i1) and carboxylate] , amines, phenols, hydroquinones and thiols.2628 The formation of photo-active charge-transfer complexes with ethylenediaminetetraacetic acid (EDTA), triethanolamine, cysteine and thiourea has also been reported.29-31 Such complex formation often gives rise to new absorption bands or to broadening of the absorbance band towards the red end of the spectrum.

The general advantages of the reaction-rate methods have been discussed.32-34 Several of these advantages are especially important with respect to diquat determination. Specificity and precision could be improved because only those species that react and cause a change in the monitored analytical signals, such as background absorbance, do not interfere. Increased specificity will make some separations unnecessary, thus providing considerable saving of time.

In this paper the development of a rate method of analysis, in which diquat forms a charge-transfer complex with cysteine, is discussed. The reaction of diquat with cysteine in basic solution is also discussed and is related to the experimental results. The rate method presented here has the additional advantages of involving more stable reagents and a simpler analytical procedure. The results of the application of the rate method to the determination of diquat in different matrices are reported.

Experimental

Apparatus

A Perkin-Elmer (Nonvalk, CT, USA) 550 SE spectro- photometer was used for recording spectra. The absorbance- time graphs were recorded with a Metrohm (Herisau, Switzerland) 662 spectrophotometer equipped with a light- guide cell and connected to a Linseis (Selb, Germany) 6512 recorder. The reaction cell used (30 cm) was cylindrical with a double-walled well through which water from a thermostat continuously flowed in order to maintain the temperature at the selected value.

Reagents

All chemicals were of analytical-reagent grade and doubly distilled water was used throughout.

A diquat stock solution (400 pg cm-3) was prepared from the pure chemical, supplied by Dr. S. Ehrenstorfer (Augs- burg, Germany) as the dibromide salt, which had previously been purified by recrystallization. Solutions of lower concen- tration were prepared by suitable dilution. A cysteine stock solution (1.21 mg ~ m - ~ ) was prepared from the Sigma (St. Louis, MO, USA) product (hydrochloride).

Recommended Procedure

The reaction cell maintained at 40 k 0.2 "C was filled with 0.8 cm3 of 4 mol dm-3 sodium hydroxide, 5 cm3 of 0.01 mol dm-3 cysteine and an appropriate volume of standard or sample diquat solution to yield a final concentration between 0.5 and 28 yg ~ m - ~ . The solution was then diluted to 10 cm3 with water, and the recorder was started in order to measure the time accurately.

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1558 Analyst, July 1994, Vol. 119

After the sample solution had been homogenized by stirring, the light-guide cell was immersed in the solution, and the absorbance (wavelength of maximum absorption 495 nm) was recorded as a function of time. The reaction rate was calculated from the absorbance-time curve obtained and the calibration graph was constructed by one of the standard methods described below.

Sample Pre-treatment

Commercial herbicides

The samples were diluted with doubly distilled water to obtain a suitable concentration level before following the recommen- ded procedure.

Water

The samples containing more than 1 pg ~ m - ~ of diquat were analysed directly. The samples containing between 0.01 and 1 yg ~ m - ~ of diquat were treated with EDTA and passed through a column of cation-exchange resin [Dowex 50- X8(Na+ form); Aldrich, Gillingham, Dorset, UK].

Diquat was retained on the resin and finally eluted with 20 cm3 of 4 mol dm-3 sodium chloride solution. The eluate was collected in a 25 cm3 calibrated flask. An appropriate aliquot of this solution was analysed by the recommended procedure.

Potatoes and soils

The procedure used for the extraction of diquat from potatoes and soils has been described previously.7Jl The herbicide was determined directly in the solution obtained from the treat- ment of the sample.

Results and Discussion

Diquut-Cysteine Charge-transfer Complex

When aqueous solutions of diquat (as the bromide salt) and cysteine are mixed in an alkaline medium, a red colour is developed. The absorption spectrum of the mixture is different from those of both components and that of the coloured free-radical ion (Fig. 1) and, hence, must be of the charge-transfer type.

The extent to which the absorption spectrum of the complex is red shifted relative to the absorption bands of the uncomplexed donor and acceptor reflects the lowering of the energy of the system due to complex formation. The presence

1.5 c I

of double charge-transfer bands is due to electron transitions from two donor energy levels or at two acceptor energy levels .35

Next, the relationship between the pH of the solution and the formation of the red complex was examined. As this complex can only be formed in alkaline medium, the concentration of sodium hydroxide was examined between 0.01 and 1.0 mol dm-3. Fig. 2 shows that the formation of the charge-transfer complex increased with increasing sodium hydroxide concentration up to about 0.3 mol dm-3 and decreased with subsequent increases in sodium hydroxide concentration.

The stability of the charge-transfer complex depended on both the cysteine and the sodium hydroxide concentrations. Fig. 3 shows the time course of the absorbance at 495 nm for

0.2 0 0.5 1 .o

[NaOHymol dm4 Fig. 2 Effect of sodium hydroxide concentration on the formation of charge-transfer complex. Conditions: [DQ*+] = 2 x mol dm-3, [cysteine] = 3 x 10-3 mol dm-3.

0.8

0.8

0.4

E 0.2 C

m Q) d c. Q n l I I

300 400 500 600 WavelengtNnm

Fig. 1 Absorption spectra of aqueous solutions: A, 5 X mol dm-3 diquat at pH 13; B, 0.005 mol dm-3 cysteine at pH 13; C, 5 X rnol dm-3 diquat and 0.005 mol dm-3 cysteine at pH 13; D, 2 X mol dm-3 diquat radical (obtained by reduction of diquat with alkaline sodium dithionite).

I I I

0 25 50 Timdmin

Fig. 3 Effect of cysteine concentration on the stability of the charge- transfer complex. Sodium hydroxide concentration: (a) 0.3 mol dm-3; (b ) 0.1 mol dm-3. A , B, C and D indicate 2 , 3 , 4 and 5 X

mol dm-3 of cysteine, respectively.

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Analyst, July 1994, Vol. 11 9 1559

several concentrations of cysteine at two levels of sodium hydroxide concentration.

The composition of the charge-transfer complex was determined from the continuous variation data shown in Fig. 4. By using -0.37 and +0.25 V (versus the NHE) as the standard redox potentials for diquat24 and cysteine (at pH 2),36 respectively, it can readily be shown that the concentration of the green free-radical ion is negligible in all of the solutions used for these studies. Consequently, it can be concluded from the data in Fig. 4 that 1 rnol of cysteine

0.8 .

0.4

0 0.5 [ Diquat~diquat]+[cysteine]

Fig. 4 Continuous variation data on the formation of a charge- transfer complex between diquat and cysteine at pH 13. Total stoichiometric concentration of diquat and cysteine = 0.01 rnol dm-3. Total ionic strength was maintained at the constant value of 0.2 by the addition of sodium chloride. A is absorbance and A. is the absorbance of the components in the absence of complex formation. Solid and broken lines represent absorbances at 495 and 350 nm, respectively

Table 1 Tolerance of different species in the determination of 3 pg ~ m - ~ of diquat

Species added NO3-, C1- , C104-, Mo042-, V03- ,

K+, Na+, NH4+ Mg2+, Ba2+, Zn2+ Ni2+, Co2+, Mn2+, Cd2+ Difenzoquat Hg2+ Paraquat

Tolerance ratio (m/m)

500" 200+ 50t 50* 207 20

Ca2+,

* Maximum ratio tested. t In the presence of 0.04 rnol dm-3 of EDTA (disodium salt).

combines with 1 rnol of diquat. Further confirmation of a 1 : 1 stoichiometry in solution comes from fulfilling the equation developed by Benesi and Hildebrand.37

[DQ'+]dAAx = l /(~xb) + l/(Keq~xb[Cyst]o) where [DQIo, E ~ , Keq, [Cystlo, b and AAx denote the concentration of diquat, molar absorptivity of the complex, equilibrium constant, concentration of cysteine, path length and the change in solution absorbance, respectively.

Treatment of the absorbance data at 495 nm for the DQ2+/ Cyst system as a function of cysteine concentration (between 0.7 and 4 x lo-' rnol dm-3) with [DQ2+] = 3 x mol dm-3 leads to a linear plot ( r = 0.998), yielding the value E = 2.2 X 104 dm3 mol-l cm-l and Keg = 5.1 dm3 mol-*.

Kinetic Aspects of the Reaction

In order to study the kinetics of the reaction of diquat with cysteine and to optimize the conditions for the analytical procedure, the effect of each reactant on the reaction rate was studied. The basicity was kept constant at 0.3 rnol dm-3 sodium hydroxide in order to reach the greatest rate formation and the greatest stability of the charge-transfer complex over a large concentration range of cysteine (see Figs. 2 and 3).

Effect of Cysteine Concentration

A linear dependence of the initial reaction rate on cysteine concentration was observed in the concentration range (0.3-6) x mol dm-3. An experimental reaction order of 0.98 f 0.01 was found. For the routine procedure, a final concentra- tion of 5 x mol dm-3 was chosen.

Effect of Diquut Concentration

There is a linear relationship between the initial reaction rate and diquat concentration up to about 8 x rnol dm-3. A reaction order of 0.99 & 0.01 was found.

The rate for formation of the charge-transfer complex is given by the equation:

From the results obtained, a kexp value of 0.96 k 0.02 dm3 mol-l s-l at 40 "C was calculated.

Table 2 Determination of diquat in real samples

Sample Diquat added* Commercial herbicide-

Reglone 200 mg cm-3 Wuters-

Sample 1 2.0 mg dm-3 Sample 2 20.0 pg dm-3

Sample 1 2.0mg kg-' Sample 2 5.0 mg kg-'

Sample 1 0.20 mg kg-' Sample 2 2.0mg kg-l

Soils-

Potatoes-

Diquat foundt

Spectrofluorimetric Kinetic method method

198.3 f 0.4 mg ~ m - ~ 200.3 f 0.3 m g ~ m - ~

1.97 f 0.07 mg dm-3 1.98 f 0.04 mg dm-3 19.5 f 0.3 pg dm-3 19.7 f 0.1 pg dm-3

1.87 k 0.06 mg kg- 4.60 f 0.14 mg kg-I

0.17 f 0.02 mg kg- 1.82 f 0.13 mg kg-1

1.94 f 0.07 mg kg-1 4.77 f 0.09 mg kg-I

0.18 f 0.1 mg kg-I 1.90 f 0.04 mg kg-I

* Certified amount for commercial herbicide. t Average (fs) for four separate determinations.

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1560 Analyst, July 1994, Vol. 119

Effect of Temperature

The temperature of the cell was vaned over the range 25- 70 "C. The reaction rate increased with increasing tempera- ture up to about 45°C and decreased with subsequent increases in temperature. This could be an over-all result of the two opposing effects that temperature has on this reaction, i .e., it increases the reaction rate while at the same time the stability of cysteine at high pH decreases.

This last effect is particularly noticeable at temperatures higher than 50 "C. A temperature of 40 "C was selected in order to ensure the greatest reaction rate.

Kinetic Determination of D k w t

Absorbance-time graphs were obtained for solutions contain- ing different amounts of diquat under the optimum condi- tions. Three methods were used for the determination of diquat. First, the initial rate (measured as a change in absorbance over 60 s) was plotted as a function of the diquat concentration. The resulting calibration graph was linear over the range 0.5-28 pg ~ m - ~ , and the relative standard deviation (s,) was 0.8% at the 10.0 pg ~ m - ~ level ( n = 10). Second, the absorbance was measured at a fixed time (6 min) after mixing; under these conditions, the calibration graph was linear over the range 0.7-26 pg ~ m - ~ with an s, of 1.6%. For the fixed- concentration method, the time needed to reach a pre-set absorbance (0.400) was taken as a basis; a linear range of 0.9- 22 pg cm-3 was obtained, with an s, of 1.9%.

The initial-rate method was chosen on the basis of its wider determination range and its lower s,. The calibration graph is described by the equation:

AAIAt = 0.0082 f 0.0001 + (0.0177 + 0.0002)C

where the time is measured in minutes, and the concentration (C) in pg cm-3.

Intederences

In order to apply the proposed method to the determination of diquat in real samples, the interference from some common components was examined. Solutions of diquat and each species were mixed to obtain samples containing 3 pg ~ m - ~ of diquat and up to 1.5 mg ~ m - ~ of the foreign species. The tolerance ratio of each foreign substance was taken as the largest amount yielding an error of less than +3% in the initial rate for 3 pg ~ m - ~ of diquat.

As can be seen from Table 1, the selectivity achieved with the proposed kinetic method was good, and so this method is useful for the determination of diquat in real samples.

Applications

The proposed kinetic method was applied to the determina- tion of diquat in real samples: herbicide formulations, waters, soils and potato tubers. The results shown in Table 2 are in good agreement with the amounts added in the preparation of the synthetic samples and with those obtained by a spectro- fluonmetric method.12

Conclusions This work clearly demonstrates that the red complex deve- loped between diquat and cysteine in basic solution is due to the formation of a charge-transfer complex. In the presence of a large excess of cysteine, the rate of formation of the complex is linearly dependent on the concentration of diquat. The kinetic method is very simple and is useful for the determina- tion of the herbicide in real samples.

The authors express their gratitude for the financial support from the Spanish DGICYT and Comunidad Aut6noma de Murcia [project PIB 93/09).

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Paper 31063421 Received October 25, 1993 Accepted January 20, 1994

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