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Determination of herbicides in soil samples by gas chromatography: optimization by the simplex method
Talanta 53 (2000) 367 – 377 www.elsevier.com/locate/talanta
Determination of herbicides in soil samples by gas chromatography: optimization by the simplex method M. Jesu´s Santos-Delgado *, Esther Crespo-Corral, Luis M. Polo-Dı´ez Department of Analytical Chemistry, Faculty of Chemistry, Complutense Uni6ersity of Madrid, 28040 Madrid, Spain Received 18 February 2000; received in revised form 3 July 2000; accepted 5 July 2000
Abstract An analytic method for the determination of phenoxy acid herbicides and 2,4-D esters in soil samples by GC-FID is described. The esterification reaction with MeOH and H2SO4 as catalyst has been used, optimizing experimental variables by the ‘Simplex method’. The recoveries in soil samples were between 76 and 97% with relative S.D.s between 4 and 7% (n =4) at level of concentration of 5 and 10 mg ml − 1 for phenoxy acids and 2,4-D esters, respectively. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Phenoxy acids; Simplex method; GC/FID
1. Introduction Phenoxy acids are used in agriculture as selective herbicides. They are usually applied as esters and salts [1], mainly on crops of cereals, such as wheat and barley, in the post-emergence phase. The determination of phenoxy acids in soils is of concern because they have harmful effects on the microflora of the soil when they are not degraded quickly enough. Moreover, because of their high solubility in water, they can be present in underground water [2]. The high polarity and low volatility of these compounds make a previous derivatization reac* Corresponding author. Tel.: +34-91-3944323; fax: + 3491-3944329. E-mail address: [email protected] (M.J. SantosDelgado).
tion necessary in order to produce more volatile compounds for their analysis by gas chromatography (GC) [3]. There are several esterification reactions based on different reagents [3–11]. Method 8150 proposed by the EPA for the determination of chlorine herbicides uses diazomethane as the derivatization reagent [12]. Diazomethane is carcinogenic and can explode under certain conditions. Therefore, it is obviously of interest to optimize safer and less toxic derivatization reactions [13]. When several experimental variables are involved, as is the case of derivatization reactions, methods of experiment design should be used. The ‘Simplex method’ (autodirected optimization method) is often used for multivariant analysis [14–16]; its advantages over the univariant method include decreasing the number of experi-
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ments to obtain optimum conditions and the time employed in them and it is possible to find out if there are interactions between the different variables. In this work a method for the determination of phenoxy acid herbicides and 2,4-D esters in soil samples (extracted with diethyl ether/H2O solvent mixture) [17] by GC-FID is proposed. The phenoxy acids were derivatized using a non-toxic reagent by means of a rapid and easy reaction with MeOH using H2SO4 as catalyst [13]; the optimum conditions were obtained by means of the autodirected optimization Simplex method. The confirmation of the presence of these analytes in the sample was carried out by means of mass spectrometry (GC-MS).
2. Experimental
2.1. Apparatus and conditions 2.1.1. GC-FID system We used a Hewlett-Packard model 5890 gas chromatograph equipped with a flame ionization detector and an HP-5 semicapillary column (30 m×0.53 mm × 2.65 mm) and a splitless injector. The data intake was carried out with PC HewlettPackard vectra Chemistation software. The temperature program for the determination of phenoxy acids was: injector temperature, 250°C; detector temperature, 280°C; oven temperature, 70°C; with gradient 70°C (2 min.) – (20°C min − 1); 150°C (4 min) – (10°C min − 1); 180°C (2 min.)– (35°C min − 1); 225°C. For the determination of 2,4-D esters the following program was used: injector temperature, 250°C; detector temperature, 280°C; oven temperature, 150°C, with gradient: 150°C (3 min) – (25°C min − 1); 190°C (1 min)–(15°C min − 1); 220°C. The carrier gas N2 flow rate was 10 – 12 ml min − 1 and the injection volume was 0.5ml. 2.1.2. GC-MS system We used a Hewlett-Packard model 5989-A gas chromatograph equipped with a split-splitless injector, injection volume 0.5 ml, and a MFE-75 capillary column (25 m ×0.25 mm ×0.25 mm).
Mass spectrophotometer with quadrupole filter (USA) HP 5989 A, Wiley library HP 59943 B integrator. The carrier gas was He. The temperature program was the same one used in GC-FID. MS measurements were performed with electron impact ionization (EI) at 70 eV and the scanned MS range was 50–320 m/z. TIC mode data was collected between 3.0 and 20.0 min. Digital scale (AND FA-2000), water thermostatic bath (Selecta), water purification system MilliQ (Millipore), vortex (selecta), rotary evaporator (Barna-Vacio), mechanic shaking system (Griffin) and 12 ml screwed tubes of (16× 0.9 cm) were used.
2.2. Reagents Methanol, hexane, acetonitrile, ethyl ether, ethyl acetate, HPLC grade (Tecknokroma), potassium chloride 95% (Panreac), sulfuric acid 98% (Panreac), methyl heptanodecanoate 99.0% (Sigma), 2,4-dichlororphenoxy-acetic acid (2,4 D) 98% and 2-(2,4,5-trichlorophenoxy)propionic acid (2,4,5-TP) 97% (Aldrich Chemie), 4-(2,4dichlorophenoxy)butyric acid, (2,4-DB), 2-(2,4dichloro-phenoxy)propionic acid (2,4-DP), (4-chloro-2-methylphenoxy)acetic acid (MCPA), 2-(4-chloro-2-methylphenoxy)pro-pionic acid (MCPP) 99.0%, and 4-(4-chloro-2-methylphenoxy)butyric acid (MCPB) 98.0% (Chem Service), 2,4-D-methylester and 2,4-D-isobutylester 99.0% (EPA), water MilliQ grade (Millipore).
2.3. Samples Two soil samples given by the Institute of Environmental Sciences of the CSIC from their estate ‘La Higueruela’ (Toledo) were studied. The composition of these soils was: Soil 1, sand 67.5%, silt 20.1%, clay 12.4%, pH (H2O) 5.4, pH (KCl) 4.2, organic matter 0.7%, electric conductivity (25°C) 3.7 mmhs × 10 − 5 cm − 1, capacity of change 9.5 cmol kg − 1 and saturation 70.0%; and Soil 2, sand 75.4%, silt 20.3%, clay 4.3%, pH (H2O) 6.0, pH (KCl) 4.8, organic matter 0.4%, electric conductivity (25°C) 3.3 mmhs× 10 − 5 cm − 1, capacity of change 8.0 cmol kg − 1 and saturation 30.0%.
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2.4. Determination of phenoxy acid herbicides and 2,4 -D esters in soil samples 2.4.1. Preparation of the sample About 5 g of soil, previously passed through a 0.25 mm sieve, was weighed and fortified with 5 mg ml − 1 phenoxy acids or 10 mg ml 2,4-D esters mixture standard solutions. Then 100 ml ethylether:water (90:10 v/v) solvent was added and shaken mechanically for 10 min. It was filtered in a vacuum through a Whatman No 1 filter paper funnel into a Buchner flask and the extracts were evaporated to dryness at 50°C on a rotary evaporator. The residue was redissolved in about 1 ml methanol [18]. 2.4.2. Deri6atization method The derivatization reaction was carried out under the following conditions: 1ml methanolic extract obtained in a previous stage was poured into a 12 ml screwed tube (16×0.9 cm), and 250 ml concentrated H2SO4 was added very gently. It was then shaken in vortex for 20 s and set aside for 12 min in a water bath at 59°C. Next, 6 ml 2% KCl solution was added. The esters were extracted in 224 ml hexane. 2.4.3. Calibration A calibration of acid herbicides in soil samples was performed at a concentration level of 0.5–5 mg ml − 1, after their esterification reaction. To do this 5 ml of 1000 mg ml − 1 internal standard solution (methyl heptanodecanoate) was added. The mixture was shaken in vortex for 1.8 min, the hexane layer was separated and a 0.5 ml aliquot was injected into the gas chromatograph, applying Table 1 Parameter values necessary for the construction of the Simplexa Variables
T (°C) tm (min) V (ml) ts (min) a
Minimum (X1) 21 5 0.2 0.5
Scale factor: E=3.
Origin (Xo) 60 10 0.5 1.0
Maximum (X2) 100 15 1.0 2.0
369
the temperature program described for phenoxy acids. The 2,4-D esters calibration was performed over the range 0.5–10 mg ml − 1. A 0.5 ml aliquot of the methanolic extract, obtained as indicated in the stage of sample preparation, was injected, and the aforementioned temperature program for the 2,4-D esters was applied.
2.5. Simplex method The four variables optimized in the esterification reaction with MeOH using H2SO4 as catalyst [19] applying the Simplex method were methylation temperature and time, hexane volume and shaking time; symbols and units, the initial value of these, as well as minimum and maximum values between which these variables can move, and the value of scale factor ‘E’, were introduced into the computer program that provides the experimental conditions. All these values are shown in Table 1. Once these data had been introduced the initial experimental conditions for five experiments (number of variables+ 1) were generated by the program. These experiments were performed and taking the area ratio (peak area or derivatized analyte/peak are of internal standard) as an evaluation criterium the set of experimental conditions that provided the worst results was rejected. Immediately the program generated experimental conditions for the new experiment, number 6. This experiment was carried out and again the experiment of the new set that provided the worst results was rejected. This process was repeated until two consecutive experiments did not differ significantly.
3. Results and discussion
3.1. Optimization of the deri6atization reaction by means of the Simplex method The Simplex method was applied to optimize the most important variables involved in the reaction of the esterification of phenoxy acids with standard methanolic solutions MeOH/H2SO4,
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Table 2 Values of the variables provided by the initial Simplex No. experiment
T (°C)
tm (min)
V (ml)
ts (min)
1 2 3 4 5
60 84 66 66 66
10.0 10.7 13.1 10.7 10.7
1.000 0.560 0.560 0.750 0.560
1.0 1.1 1.1 1.1 1.5
which according to the literature [13] and previous analysis [20] were: methylation temperature (T), methylation time (tm), hexane volume (V) and shaking time (ts). The computer programme was used based on the resolution of a matrix of K dimensions (K = number of experimental variables, in this case four). Table 2 shows the matrix for the called physical variables provided by the initial simplex for each vertex of the Simplex. These values were calculated starting from Eq. (1) whereby Xphy is the physical value of the variable X (any experimental variable), Xmat is the corresponding mathematical coordinate; Xo is the value of origin for each variable and X1 and X2 the minimum and maximum values, respectively, for each variable. ‘E’ is the factor of scale, a numerical value that will determine the size of the Simplex. Xphys =Xo +Xmat ×
X2 −X1 . E
(1)
Once the experiments had been performed with the conditions Xphy, the vertex of worst response, experiment number 1, was rejected. Next, a new symmetrical vertex with respect to the hyperpolyhedron formed by the rest of the vertises was obtained, and this is given by Eq. (2): V*= Vc + (Vc −Vi) i
(2)
where the test vertex is represented by Vi (Xi1, Xi2, Xi3,…Xin ), V *i is its symmetrical vertex and Vc the centroide of the retained vertices. In Table 3 the results obtained by GC-FID for the variables studied until the end of the optimization are shown. Table 3 indicates the number of the experiment, the values of its physical coordinates, relative areas for each phenoxy acid herbicide,
and the number of the experiment that was rejected as well as the experiments retained in each case. The experimental conditions of experiments 10, 12 and 20 were outside the experimental domain, therefore, these experiments were not carried out, and were considered as giving the worst response. The evolution process of the Simplex method was repeated until the results of two consecutive experiments did not significantly differ, as can be seen in the representation of the Simplex evolution for each compound (Fig. 1) experiments numbers 18, 19 and 21. The experiment that provided the best results was number 13, which gave the following optimum values for the variables of the derivatization process: T= 59°C, tm = 12.0 min, V= 224 ml, and ts = 1.8 min. In experiments 14–17 there was a movement away from the optimum conditions, which may be due to a rotation brought about by the geometry of the movement. In the process of the Simplex a net increase in the area ratio for all compounds was observed, whose variation was less in proximity to the optimum. The optimum conditions obtained (experiment number 13) and the use of a screwed tube of small internal diameter, 0.9 cm, permitted a noticeable reduction, by a factor of 10 approximately, in the volume of the extraction solvent, hexane, after its methylation. Moreover, the volume of reagents involved in the derivatization reaction decreased, which led to a notable increase in the sensitivity of the method, a reduced consumption of reagents and a reduced generation of residuals with respect to the bibliographic method applied to other matrices [13].
3.2. Analysis of soil samples Optimum experimental conditions were applied to the analysis of two soil samples from the experimental estate ‘La Higueruela’ (Toledo). These soils were both of franc-sandy texture, with a low clay content, very poor in organic matter and with quite an acid pH. However, soil 1 had almost twice as much clay and organic matter as soil 2.
Exp. retained
2, 3, 4, 5 2, 3, 5, 6 3, 5, 6, 7 5, 6, 7, 8 6, 7, 8, 9 6, 7, 8, 9 6, 7, 8, 9 6, 7, 8, 9 6, 7, 9, 13 6, 7, 13 14 6, 7, 13 14 6, 7, 13, 15 6, 13, 16, 17 6, 13, 16, 18 6, 13, 18, 19 6, 13, 18, 19 13,18, 19, 21
Exp No.
1 2 3 4 5 6 7 8 9 10* 11 12* 13 14 15 16 17 18 19 20* 21 22 1 4 2 3 5 10 11 12 8 9 15 16 7 17 16 20 6
Exp. rejected 60 84 66 66 66 72 47 57 61 53 66 53 59 63 59 56 58 75 73 83 80 44
T (°C)
Table 3 Evolution and results of the Simplex optimization method
10.0 10.7 13.1 10.7 10.7 11.5 11.9 9.0 11.6 11.3 10.7 11.3 12.0 14.6 13.4 9.9 9.3 10.5 12.2 12.7 12.6 13.0
tm (min) 1.000 0.560 0.560 0.750 0.560 0.340 0.420 0.351 0.335 0.165 0.558 0.165 0.224 0.308 0.311 0.339 0.351 0.207 0.205 0.148 0.251 0.327
V (ml) 1.0 1.1 1.1 1.1 1.5 1.2 1.3 1.4 1.7 1.3 1.5 1.38 1.8 1.6 1.3 1.2 1.5 1.5 1.4 1.8 1.6 1.8
ts (min) 0.458 0.521 0.523 0.498 0.576 0.636 0.623 0.582 0.586 – 0.513 – 0.709 0.600 0.622 0.634 0.628 0.656. 0.674 – 0.653 –
MCPP 0.377 0.411 0.414 0.386 0.445 0.519 0.513 0.460 0.461 – 0.425 – 0.529 0.455 0.481 0.506 0.480 0.515 0.503 – 0.509 –
MCPA 0.396 0.422 0.434 0.405 0.479 0.5444 0.524 0.480 0.481 – 0.435 – 0.581 0.487 0.516 0.523 0.541 0.553 0.552 – 0.545 –
2,4-DP
0.291 0.321 0.304 0.283 0.340 0.406 0.394 0.329 0.357 – 0.322 – 0.414 0.369 0.376 0.380 0.402 0.345 0.386 – 0.388 –
2,4-D
0.390 0.458 0.445 0.397 0.456 0.508 0.509 0.437 0.508 – 0.416 – 0.587 0.520 0.575 0.539 0.521 0.579 0.580 – 0.515 –
2,4,5-TP
0.494 0.550 0.572 0.500 0.626 0.749 0.716 0.655 0.729 – 0.595 – 0.890 0.772 0.850 0.819 0.746 0.831 0.865 – 0.807 –
MCPB
0.624 0.672 0.749 0.637 0.803 0.853 0.941 0.806 0.980 – 0.673 – 0.994 0.985 0.972 0.956 0.889 0.985 0.988 – 0.973 –
2,4-DB
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First, an analysis of the unenriched soil samples was carried out by GC-FID, verifying the absence of herbicides. The soil samples were then fortified with a solution containing a mixture of the seven
phenoxy acid herbicides in the concentration range 0.5–5 mg ml − 1 for each compound, or with a solution of 2,4-D esters in the concentration range 0.5–10 mg ml − 1. The herbicides of the
Fig. 1. Evolution of the sign for each analyte upon applying the Simplex method.
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373
Fig. 2. Chromatograms of the soil 1 sample by GC/MS: column MFE-75 (25 m × 0.25 mm×0.25 mm) (a) for phenoxy acid herbicides mixture (1-MCPP, 2-MCPA, 3-2,4-DP, 4-2,4-D, 5-2,4,5-TP, 6-MCPB, 7-2,4-DB and 8-C17) and (b) for 2,4-D esters mixture (1-2,4-Dmethyl ester and 2-2,4-Disobutyl ester).
fortified samples were identified by GC-MS obtaining similar chromatograms for the two types of soil. In Fig. 2 the chromatograms for soil 1 are shown. The identification of each herbicide was performed by means of their library GC-MS. Variables to optimize the extraction of the herbicides from the soil, solvent extract (acetonitrile, ethyl acetate and ethyl ether), organic solvent:water ratio (85:15 – 95:5), shaking time (5–15 min) and also in the case of the phenoxy acid herbicides the effect of the pH (3.3 – 4.3) were determined in fortified samples. The best results obtained from this study for both phenoxy acids and 2,4-D esters were: ethyl ether:water ratio (90:10 v/v) with a shaking time of 10 min and 3.8 pH for phenoxy acids. The chromatograms for soils 1 and 2 are shown in Figs. 3 and 4.
The analysis of the soil samples performed by GC-FID allowed the determination of the analytic characteristics and recovery values that are shown in Tables 4 and 5 for soils 1 and 2, respectively. The recoveries from soil 2 (85–97%) were always greater than from soil 1 (76–90%), possibly due to the different percentage of colloids both mineral and organic. The limits of detection, LODs, obtained for the phenoxy acids were superior at 27 mg l − 1, and at 75 mg l − 1 for the 2,4-D esters. One can observe that the most sensitive analyte in the case of phenoxy acids was MCPB, and for 2,4-D esters, isobutylester. The R.S.D., obtained at a concentration level of 5 mg ml − 1 and four measurements was 4–7% for the phenoxy acids; and for 2,4-D esters at a concentration level of 10 mg ml − 1 it was 5–7%.
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4. Conclusions A method for the determination of phenoxy acid herbicides and 2,4-D esters by GC-FID/MS in soil samples has been developed. The derivatization of the phenoxy acid herbicides with MeOH and H2SO4 as catalyst was satisfactory, the proposed method being simple, quick and of low toxicity.
The variables of the derivatization reaction were optimized by means of the autodirected multivariable Simplex method leading to a decrease in reagents and shaking time which reduces costs and the generation of residuals. Optimization was achieved with the minimum number of experiments, therefore, experimentation time was decreased with respect to univariant methods.
Fig. 3. Chromatograms obtained by GC/FID (column HP-5 (30 m ×0.53 mm ×2.65 mm) of soil 1 sample enriched with: (a) 5 mg ml − 1 of phenoxy acid herbicides mixture and (b) 10 mg ml − 1 of 2,4-D esters mixture. Compounds enumerated as in Fig. 2.
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375
Fig. 4. Chromatograms obtained by GC/FID column HP-5 (30 m × 0.53 mm× 2.65 m) of the soil 2 sample enriched with: (a) 5 mg ml − 1 of phenoxy acid herbicides mixture and (b) 10 mg ml − 1 of 2,4-D esters mixture. Compounds enumerated as in Fig. 2.
The proposed method is valid for carrying out the determination of phenoxy acid herbicides and 2,4-D esters in soil samples in the concentration range 0.5–10 mg l − 1, allowing the detection of (6–26)* and (17 – 21) mg kg − 1, respectively, for each kilogram of soil sample.
Acknowledgements The financial support of the Spanish DGICYT Project PB 96-0642 and the Spectroscopy Center Mass Spectrometer Service (UCM) is gratefully acknowledged.
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Table 4 Analytic characteristics for the phenoxy acid herbicides and 2,4-D esters in soil 1 samples Sensitivity (mV mg−1 l−1)
LODa.(mg kg−1)
LOQb (mgkg−1) R.S.D.c (%)
R d (%)
Herbicides
Calibration
MCPP MCPA 2,4-DP 2,4-Disobutylester 2,4,5-TP MCPB 2,4-Disobutlester 2,4-Dmethylester 2,4-Disobutylester
y=0.081x+0.018 19.4 y= 0.062x+0.021 15.4 y= 0.067x+0.026 12.0 y=0.044x+0.008 8.3
11 14 17 26
54 70 88 132
6* 7* 6* 7*
82 79 81 78
y= 0.072x+0.030 y =0.120x+0.068 y=0.085x+0.058 y= 2048x+1801 y= 2460x+3287
10 6 10 20 17
50 30 49 101 86
5* 4* 5* 6** 5**
85 90 87 76 85
20.0 31.7 20.0 10.8 11.1
a
Limit of detection. Limit of quantification. c Relative S.D. d Recovery. * R.S.D. for n=4y [C] =5 mg ml−1. ** R.S.D. for n=4y [C] = 10 mg ml−1. b
Table 5 Analytic characteristics for the phenoxy acid herbicides and 2,4-D esters in soil 2 samples Herbicides
Calibration
Sensitivity (mV mg−1 l−1)
MCPP MCPA 2,4-DP 2,4-D 2,4,5-TP MCPB 2,4-DB 2,4-Dmethylester 2,4-Disobutylester
y=0.113x+0.010 y=0.108x+0.048 y=0.100x+0.047 y=0.073x+0.031 y=0.102x+0.069 y=0.137x+0.142 y=0.111x+0.115 y=2628x+3222 y=3220x+3577
24.3 18.6 14.8 10.0 24.6 33.1 22.3 11.4 12.8
LODa (mg kg−1) 9 12 15 23 9 6 10 21 18
LOQb (mg kg−1) 48 60 77 118 47 36 51 104 90
RSDc (%) 5* 6* 5* 7* 4* 4* 4* 7** 7**
Rd (%) 92 90 88 85 91 95 87 92 97
a
Limit of detection. Limit of quantification. c Relative S.D. d Recovery. * R.S.D. for n=4y [C] =5 mg ml−1. ** R.S.D. for n=4y [C] = 10 mg ml−1. b
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Determination of herbicides in soil samples by gas chromatography: optimization by the simplex method M. Jesu´s Santos-Delgado *, Esther Crespo-Corral, Luis M. Polo-Dı´ez Department of Analytical Chemistry, Faculty of Chemistry, Complutense Uni6ersity of Madrid, 28040 Madrid, Spain Received 18 February 2000; received in revised form 3 July 2000; accepted 5 July 2000
Abstract An analytic method for the determination of phenoxy acid herbicides and 2,4-D esters in soil samples by GC-FID is described. The esterification reaction with MeOH and H2SO4 as catalyst has been used, optimizing experimental variables by the ‘Simplex method’. The recoveries in soil samples were between 76 and 97% with relative S.D.s between 4 and 7% (n =4) at level of concentration of 5 and 10 mg ml − 1 for phenoxy acids and 2,4-D esters, respectively. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Phenoxy acids; Simplex method; GC/FID
1. Introduction Phenoxy acids are used in agriculture as selective herbicides. They are usually applied as esters and salts [1], mainly on crops of cereals, such as wheat and barley, in the post-emergence phase. The determination of phenoxy acids in soils is of concern because they have harmful effects on the microflora of the soil when they are not degraded quickly enough. Moreover, because of their high solubility in water, they can be present in underground water [2]. The high polarity and low volatility of these compounds make a previous derivatization reac* Corresponding author. Tel.: +34-91-3944323; fax: + 3491-3944329. E-mail address: [email protected] (M.J. SantosDelgado).
tion necessary in order to produce more volatile compounds for their analysis by gas chromatography (GC) [3]. There are several esterification reactions based on different reagents [3–11]. Method 8150 proposed by the EPA for the determination of chlorine herbicides uses diazomethane as the derivatization reagent [12]. Diazomethane is carcinogenic and can explode under certain conditions. Therefore, it is obviously of interest to optimize safer and less toxic derivatization reactions [13]. When several experimental variables are involved, as is the case of derivatization reactions, methods of experiment design should be used. The ‘Simplex method’ (autodirected optimization method) is often used for multivariant analysis [14–16]; its advantages over the univariant method include decreasing the number of experi-
0039-9140/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 0 0 ) 0 0 4 9 8 - 7
368
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ments to obtain optimum conditions and the time employed in them and it is possible to find out if there are interactions between the different variables. In this work a method for the determination of phenoxy acid herbicides and 2,4-D esters in soil samples (extracted with diethyl ether/H2O solvent mixture) [17] by GC-FID is proposed. The phenoxy acids were derivatized using a non-toxic reagent by means of a rapid and easy reaction with MeOH using H2SO4 as catalyst [13]; the optimum conditions were obtained by means of the autodirected optimization Simplex method. The confirmation of the presence of these analytes in the sample was carried out by means of mass spectrometry (GC-MS).
2. Experimental
2.1. Apparatus and conditions 2.1.1. GC-FID system We used a Hewlett-Packard model 5890 gas chromatograph equipped with a flame ionization detector and an HP-5 semicapillary column (30 m×0.53 mm × 2.65 mm) and a splitless injector. The data intake was carried out with PC HewlettPackard vectra Chemistation software. The temperature program for the determination of phenoxy acids was: injector temperature, 250°C; detector temperature, 280°C; oven temperature, 70°C; with gradient 70°C (2 min.) – (20°C min − 1); 150°C (4 min) – (10°C min − 1); 180°C (2 min.)– (35°C min − 1); 225°C. For the determination of 2,4-D esters the following program was used: injector temperature, 250°C; detector temperature, 280°C; oven temperature, 150°C, with gradient: 150°C (3 min) – (25°C min − 1); 190°C (1 min)–(15°C min − 1); 220°C. The carrier gas N2 flow rate was 10 – 12 ml min − 1 and the injection volume was 0.5ml. 2.1.2. GC-MS system We used a Hewlett-Packard model 5989-A gas chromatograph equipped with a split-splitless injector, injection volume 0.5 ml, and a MFE-75 capillary column (25 m ×0.25 mm ×0.25 mm).
Mass spectrophotometer with quadrupole filter (USA) HP 5989 A, Wiley library HP 59943 B integrator. The carrier gas was He. The temperature program was the same one used in GC-FID. MS measurements were performed with electron impact ionization (EI) at 70 eV and the scanned MS range was 50–320 m/z. TIC mode data was collected between 3.0 and 20.0 min. Digital scale (AND FA-2000), water thermostatic bath (Selecta), water purification system MilliQ (Millipore), vortex (selecta), rotary evaporator (Barna-Vacio), mechanic shaking system (Griffin) and 12 ml screwed tubes of (16× 0.9 cm) were used.
2.2. Reagents Methanol, hexane, acetonitrile, ethyl ether, ethyl acetate, HPLC grade (Tecknokroma), potassium chloride 95% (Panreac), sulfuric acid 98% (Panreac), methyl heptanodecanoate 99.0% (Sigma), 2,4-dichlororphenoxy-acetic acid (2,4 D) 98% and 2-(2,4,5-trichlorophenoxy)propionic acid (2,4,5-TP) 97% (Aldrich Chemie), 4-(2,4dichlorophenoxy)butyric acid, (2,4-DB), 2-(2,4dichloro-phenoxy)propionic acid (2,4-DP), (4-chloro-2-methylphenoxy)acetic acid (MCPA), 2-(4-chloro-2-methylphenoxy)pro-pionic acid (MCPP) 99.0%, and 4-(4-chloro-2-methylphenoxy)butyric acid (MCPB) 98.0% (Chem Service), 2,4-D-methylester and 2,4-D-isobutylester 99.0% (EPA), water MilliQ grade (Millipore).
2.3. Samples Two soil samples given by the Institute of Environmental Sciences of the CSIC from their estate ‘La Higueruela’ (Toledo) were studied. The composition of these soils was: Soil 1, sand 67.5%, silt 20.1%, clay 12.4%, pH (H2O) 5.4, pH (KCl) 4.2, organic matter 0.7%, electric conductivity (25°C) 3.7 mmhs × 10 − 5 cm − 1, capacity of change 9.5 cmol kg − 1 and saturation 70.0%; and Soil 2, sand 75.4%, silt 20.3%, clay 4.3%, pH (H2O) 6.0, pH (KCl) 4.8, organic matter 0.4%, electric conductivity (25°C) 3.3 mmhs× 10 − 5 cm − 1, capacity of change 8.0 cmol kg − 1 and saturation 30.0%.
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2.4. Determination of phenoxy acid herbicides and 2,4 -D esters in soil samples 2.4.1. Preparation of the sample About 5 g of soil, previously passed through a 0.25 mm sieve, was weighed and fortified with 5 mg ml − 1 phenoxy acids or 10 mg ml 2,4-D esters mixture standard solutions. Then 100 ml ethylether:water (90:10 v/v) solvent was added and shaken mechanically for 10 min. It was filtered in a vacuum through a Whatman No 1 filter paper funnel into a Buchner flask and the extracts were evaporated to dryness at 50°C on a rotary evaporator. The residue was redissolved in about 1 ml methanol [18]. 2.4.2. Deri6atization method The derivatization reaction was carried out under the following conditions: 1ml methanolic extract obtained in a previous stage was poured into a 12 ml screwed tube (16×0.9 cm), and 250 ml concentrated H2SO4 was added very gently. It was then shaken in vortex for 20 s and set aside for 12 min in a water bath at 59°C. Next, 6 ml 2% KCl solution was added. The esters were extracted in 224 ml hexane. 2.4.3. Calibration A calibration of acid herbicides in soil samples was performed at a concentration level of 0.5–5 mg ml − 1, after their esterification reaction. To do this 5 ml of 1000 mg ml − 1 internal standard solution (methyl heptanodecanoate) was added. The mixture was shaken in vortex for 1.8 min, the hexane layer was separated and a 0.5 ml aliquot was injected into the gas chromatograph, applying Table 1 Parameter values necessary for the construction of the Simplexa Variables
T (°C) tm (min) V (ml) ts (min) a
Minimum (X1) 21 5 0.2 0.5
Scale factor: E=3.
Origin (Xo) 60 10 0.5 1.0
Maximum (X2) 100 15 1.0 2.0
369
the temperature program described for phenoxy acids. The 2,4-D esters calibration was performed over the range 0.5–10 mg ml − 1. A 0.5 ml aliquot of the methanolic extract, obtained as indicated in the stage of sample preparation, was injected, and the aforementioned temperature program for the 2,4-D esters was applied.
2.5. Simplex method The four variables optimized in the esterification reaction with MeOH using H2SO4 as catalyst [19] applying the Simplex method were methylation temperature and time, hexane volume and shaking time; symbols and units, the initial value of these, as well as minimum and maximum values between which these variables can move, and the value of scale factor ‘E’, were introduced into the computer program that provides the experimental conditions. All these values are shown in Table 1. Once these data had been introduced the initial experimental conditions for five experiments (number of variables+ 1) were generated by the program. These experiments were performed and taking the area ratio (peak area or derivatized analyte/peak are of internal standard) as an evaluation criterium the set of experimental conditions that provided the worst results was rejected. Immediately the program generated experimental conditions for the new experiment, number 6. This experiment was carried out and again the experiment of the new set that provided the worst results was rejected. This process was repeated until two consecutive experiments did not differ significantly.
3. Results and discussion
3.1. Optimization of the deri6atization reaction by means of the Simplex method The Simplex method was applied to optimize the most important variables involved in the reaction of the esterification of phenoxy acids with standard methanolic solutions MeOH/H2SO4,
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Table 2 Values of the variables provided by the initial Simplex No. experiment
T (°C)
tm (min)
V (ml)
ts (min)
1 2 3 4 5
60 84 66 66 66
10.0 10.7 13.1 10.7 10.7
1.000 0.560 0.560 0.750 0.560
1.0 1.1 1.1 1.1 1.5
which according to the literature [13] and previous analysis [20] were: methylation temperature (T), methylation time (tm), hexane volume (V) and shaking time (ts). The computer programme was used based on the resolution of a matrix of K dimensions (K = number of experimental variables, in this case four). Table 2 shows the matrix for the called physical variables provided by the initial simplex for each vertex of the Simplex. These values were calculated starting from Eq. (1) whereby Xphy is the physical value of the variable X (any experimental variable), Xmat is the corresponding mathematical coordinate; Xo is the value of origin for each variable and X1 and X2 the minimum and maximum values, respectively, for each variable. ‘E’ is the factor of scale, a numerical value that will determine the size of the Simplex. Xphys =Xo +Xmat ×
X2 −X1 . E
(1)
Once the experiments had been performed with the conditions Xphy, the vertex of worst response, experiment number 1, was rejected. Next, a new symmetrical vertex with respect to the hyperpolyhedron formed by the rest of the vertises was obtained, and this is given by Eq. (2): V*= Vc + (Vc −Vi) i
(2)
where the test vertex is represented by Vi (Xi1, Xi2, Xi3,…Xin ), V *i is its symmetrical vertex and Vc the centroide of the retained vertices. In Table 3 the results obtained by GC-FID for the variables studied until the end of the optimization are shown. Table 3 indicates the number of the experiment, the values of its physical coordinates, relative areas for each phenoxy acid herbicide,
and the number of the experiment that was rejected as well as the experiments retained in each case. The experimental conditions of experiments 10, 12 and 20 were outside the experimental domain, therefore, these experiments were not carried out, and were considered as giving the worst response. The evolution process of the Simplex method was repeated until the results of two consecutive experiments did not significantly differ, as can be seen in the representation of the Simplex evolution for each compound (Fig. 1) experiments numbers 18, 19 and 21. The experiment that provided the best results was number 13, which gave the following optimum values for the variables of the derivatization process: T= 59°C, tm = 12.0 min, V= 224 ml, and ts = 1.8 min. In experiments 14–17 there was a movement away from the optimum conditions, which may be due to a rotation brought about by the geometry of the movement. In the process of the Simplex a net increase in the area ratio for all compounds was observed, whose variation was less in proximity to the optimum. The optimum conditions obtained (experiment number 13) and the use of a screwed tube of small internal diameter, 0.9 cm, permitted a noticeable reduction, by a factor of 10 approximately, in the volume of the extraction solvent, hexane, after its methylation. Moreover, the volume of reagents involved in the derivatization reaction decreased, which led to a notable increase in the sensitivity of the method, a reduced consumption of reagents and a reduced generation of residuals with respect to the bibliographic method applied to other matrices [13].
3.2. Analysis of soil samples Optimum experimental conditions were applied to the analysis of two soil samples from the experimental estate ‘La Higueruela’ (Toledo). These soils were both of franc-sandy texture, with a low clay content, very poor in organic matter and with quite an acid pH. However, soil 1 had almost twice as much clay and organic matter as soil 2.
Exp. retained
2, 3, 4, 5 2, 3, 5, 6 3, 5, 6, 7 5, 6, 7, 8 6, 7, 8, 9 6, 7, 8, 9 6, 7, 8, 9 6, 7, 8, 9 6, 7, 9, 13 6, 7, 13 14 6, 7, 13 14 6, 7, 13, 15 6, 13, 16, 17 6, 13, 16, 18 6, 13, 18, 19 6, 13, 18, 19 13,18, 19, 21
Exp No.
1 2 3 4 5 6 7 8 9 10* 11 12* 13 14 15 16 17 18 19 20* 21 22 1 4 2 3 5 10 11 12 8 9 15 16 7 17 16 20 6
Exp. rejected 60 84 66 66 66 72 47 57 61 53 66 53 59 63 59 56 58 75 73 83 80 44
T (°C)
Table 3 Evolution and results of the Simplex optimization method
10.0 10.7 13.1 10.7 10.7 11.5 11.9 9.0 11.6 11.3 10.7 11.3 12.0 14.6 13.4 9.9 9.3 10.5 12.2 12.7 12.6 13.0
tm (min) 1.000 0.560 0.560 0.750 0.560 0.340 0.420 0.351 0.335 0.165 0.558 0.165 0.224 0.308 0.311 0.339 0.351 0.207 0.205 0.148 0.251 0.327
V (ml) 1.0 1.1 1.1 1.1 1.5 1.2 1.3 1.4 1.7 1.3 1.5 1.38 1.8 1.6 1.3 1.2 1.5 1.5 1.4 1.8 1.6 1.8
ts (min) 0.458 0.521 0.523 0.498 0.576 0.636 0.623 0.582 0.586 – 0.513 – 0.709 0.600 0.622 0.634 0.628 0.656. 0.674 – 0.653 –
MCPP 0.377 0.411 0.414 0.386 0.445 0.519 0.513 0.460 0.461 – 0.425 – 0.529 0.455 0.481 0.506 0.480 0.515 0.503 – 0.509 –
MCPA 0.396 0.422 0.434 0.405 0.479 0.5444 0.524 0.480 0.481 – 0.435 – 0.581 0.487 0.516 0.523 0.541 0.553 0.552 – 0.545 –
2,4-DP
0.291 0.321 0.304 0.283 0.340 0.406 0.394 0.329 0.357 – 0.322 – 0.414 0.369 0.376 0.380 0.402 0.345 0.386 – 0.388 –
2,4-D
0.390 0.458 0.445 0.397 0.456 0.508 0.509 0.437 0.508 – 0.416 – 0.587 0.520 0.575 0.539 0.521 0.579 0.580 – 0.515 –
2,4,5-TP
0.494 0.550 0.572 0.500 0.626 0.749 0.716 0.655 0.729 – 0.595 – 0.890 0.772 0.850 0.819 0.746 0.831 0.865 – 0.807 –
MCPB
0.624 0.672 0.749 0.637 0.803 0.853 0.941 0.806 0.980 – 0.673 – 0.994 0.985 0.972 0.956 0.889 0.985 0.988 – 0.973 –
2,4-DB
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First, an analysis of the unenriched soil samples was carried out by GC-FID, verifying the absence of herbicides. The soil samples were then fortified with a solution containing a mixture of the seven
phenoxy acid herbicides in the concentration range 0.5–5 mg ml − 1 for each compound, or with a solution of 2,4-D esters in the concentration range 0.5–10 mg ml − 1. The herbicides of the
Fig. 1. Evolution of the sign for each analyte upon applying the Simplex method.
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Fig. 2. Chromatograms of the soil 1 sample by GC/MS: column MFE-75 (25 m × 0.25 mm×0.25 mm) (a) for phenoxy acid herbicides mixture (1-MCPP, 2-MCPA, 3-2,4-DP, 4-2,4-D, 5-2,4,5-TP, 6-MCPB, 7-2,4-DB and 8-C17) and (b) for 2,4-D esters mixture (1-2,4-Dmethyl ester and 2-2,4-Disobutyl ester).
fortified samples were identified by GC-MS obtaining similar chromatograms for the two types of soil. In Fig. 2 the chromatograms for soil 1 are shown. The identification of each herbicide was performed by means of their library GC-MS. Variables to optimize the extraction of the herbicides from the soil, solvent extract (acetonitrile, ethyl acetate and ethyl ether), organic solvent:water ratio (85:15 – 95:5), shaking time (5–15 min) and also in the case of the phenoxy acid herbicides the effect of the pH (3.3 – 4.3) were determined in fortified samples. The best results obtained from this study for both phenoxy acids and 2,4-D esters were: ethyl ether:water ratio (90:10 v/v) with a shaking time of 10 min and 3.8 pH for phenoxy acids. The chromatograms for soils 1 and 2 are shown in Figs. 3 and 4.
The analysis of the soil samples performed by GC-FID allowed the determination of the analytic characteristics and recovery values that are shown in Tables 4 and 5 for soils 1 and 2, respectively. The recoveries from soil 2 (85–97%) were always greater than from soil 1 (76–90%), possibly due to the different percentage of colloids both mineral and organic. The limits of detection, LODs, obtained for the phenoxy acids were superior at 27 mg l − 1, and at 75 mg l − 1 for the 2,4-D esters. One can observe that the most sensitive analyte in the case of phenoxy acids was MCPB, and for 2,4-D esters, isobutylester. The R.S.D., obtained at a concentration level of 5 mg ml − 1 and four measurements was 4–7% for the phenoxy acids; and for 2,4-D esters at a concentration level of 10 mg ml − 1 it was 5–7%.
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4. Conclusions A method for the determination of phenoxy acid herbicides and 2,4-D esters by GC-FID/MS in soil samples has been developed. The derivatization of the phenoxy acid herbicides with MeOH and H2SO4 as catalyst was satisfactory, the proposed method being simple, quick and of low toxicity.
The variables of the derivatization reaction were optimized by means of the autodirected multivariable Simplex method leading to a decrease in reagents and shaking time which reduces costs and the generation of residuals. Optimization was achieved with the minimum number of experiments, therefore, experimentation time was decreased with respect to univariant methods.
Fig. 3. Chromatograms obtained by GC/FID (column HP-5 (30 m ×0.53 mm ×2.65 mm) of soil 1 sample enriched with: (a) 5 mg ml − 1 of phenoxy acid herbicides mixture and (b) 10 mg ml − 1 of 2,4-D esters mixture. Compounds enumerated as in Fig. 2.
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Fig. 4. Chromatograms obtained by GC/FID column HP-5 (30 m × 0.53 mm× 2.65 m) of the soil 2 sample enriched with: (a) 5 mg ml − 1 of phenoxy acid herbicides mixture and (b) 10 mg ml − 1 of 2,4-D esters mixture. Compounds enumerated as in Fig. 2.
The proposed method is valid for carrying out the determination of phenoxy acid herbicides and 2,4-D esters in soil samples in the concentration range 0.5–10 mg l − 1, allowing the detection of (6–26)* and (17 – 21) mg kg − 1, respectively, for each kilogram of soil sample.
Acknowledgements The financial support of the Spanish DGICYT Project PB 96-0642 and the Spectroscopy Center Mass Spectrometer Service (UCM) is gratefully acknowledged.
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Table 4 Analytic characteristics for the phenoxy acid herbicides and 2,4-D esters in soil 1 samples Sensitivity (mV mg−1 l−1)
LODa.(mg kg−1)
LOQb (mgkg−1) R.S.D.c (%)
R d (%)
Herbicides
Calibration
MCPP MCPA 2,4-DP 2,4-Disobutylester 2,4,5-TP MCPB 2,4-Disobutlester 2,4-Dmethylester 2,4-Disobutylester
y=0.081x+0.018 19.4 y= 0.062x+0.021 15.4 y= 0.067x+0.026 12.0 y=0.044x+0.008 8.3
11 14 17 26
54 70 88 132
6* 7* 6* 7*
82 79 81 78
y= 0.072x+0.030 y =0.120x+0.068 y=0.085x+0.058 y= 2048x+1801 y= 2460x+3287
10 6 10 20 17
50 30 49 101 86
5* 4* 5* 6** 5**
85 90 87 76 85
20.0 31.7 20.0 10.8 11.1
a
Limit of detection. Limit of quantification. c Relative S.D. d Recovery. * R.S.D. for n=4y [C] =5 mg ml−1. ** R.S.D. for n=4y [C] = 10 mg ml−1. b
Table 5 Analytic characteristics for the phenoxy acid herbicides and 2,4-D esters in soil 2 samples Herbicides
Calibration
Sensitivity (mV mg−1 l−1)
MCPP MCPA 2,4-DP 2,4-D 2,4,5-TP MCPB 2,4-DB 2,4-Dmethylester 2,4-Disobutylester
y=0.113x+0.010 y=0.108x+0.048 y=0.100x+0.047 y=0.073x+0.031 y=0.102x+0.069 y=0.137x+0.142 y=0.111x+0.115 y=2628x+3222 y=3220x+3577
24.3 18.6 14.8 10.0 24.6 33.1 22.3 11.4 12.8
LODa (mg kg−1) 9 12 15 23 9 6 10 21 18
LOQb (mg kg−1) 48 60 77 118 47 36 51 104 90
RSDc (%) 5* 6* 5* 7* 4* 4* 4* 7** 7**
Rd (%) 92 90 88 85 91 95 87 92 97
a
Limit of detection. Limit of quantification. c Relative S.D. d Recovery. * R.S.D. for n=4y [C] =5 mg ml−1. ** R.S.D. for n=4y [C] = 10 mg ml−1. b
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