69 8
Ele~crophore.~it 1992.
C . Desiderio and S . Fanali
Claudia Desiderio Salvatore Fanali Istituto di Cromatografia del C.N.R. Monterotondo Scalo, Roma
13,698-700
Atrazine and simazine determination in river water samples by micellar electrokinetic capillary chromatography A rapid and sensitive micellar electrokinetic capillary chromatography analytical method was used for the determination of chlorotriazine herbicides in river water samples. Several electrolyte systems in the pH range 7-10 were tested in order to optimize the separation. The two compounds were separated in less than 10 min and the determination limit was about 0.4 ppb for each herbicide. Kecovery values of the method were in the range 8O0/o-117%.
1 Introduction S-triazine herbicides are among the most widely used pesticides to control broadleaf and grassy weeds in corn and other crops [l]. Due to their extensive use and their relatively high persistence, chlorotriazines contaminate the acquatic environment through agricultural runoff, direct application and leaching into ground water, in concentrations that are increasing [2]. Many efforts have been devoted to develop rapid ascays for the determination of triazine herbicides at low levels in water. Several analytical techniques that have been used so far for the determination of these compounds are gas-chromatography (GC) [3-61, high performance liquid chromatography (HPLC) [2, 5, 7, 81, thin layer chromatography (TLC) [9], isotachophoresis (ITP) [10],and capillary zone electrophoresis (CZE) [ 113.Micellar electrokinetic capillary chromatography (MECC), first introduced by Terabe and co-workers [12, 131, is a relatively new type of separation method, combining electrophoresis and chromatography [14], which is mainly suited for the resolution of neutral compounds. In M E C C an ionic surfactant is added to the background electrolyte (BGE) at a concentration level above its critical micellar concentration. When a high vollage is applied to the system, the uncharged compounds, moving with the same velocity as that of the electroosmotic flow, will separate by different partitioning between the aqueous phase and the micellar one. The last phase moves in the opposite direction to the one before. This paper presents our investigation on the possibility of applying this new electrophoretic technique to the analysis of atrazine and simazine in river water samples.
2 Materials and methods 2.1 Apparatus Electrophoretic experiments were performed in a laboratory-made apparatus consisting of a modified Varian UV 2550 detector and a power supply (Glassman LH60R) able to deliver either constant voltage or constant current. A fused silica capillary tube (50 cm long X 75 pm ID, 36 cm to
Correspondence: Dr. S. Fanali,Istituto di Cromatografia del C.N.R.,Area della Ricerca di Roma, P.O. Box 10,I-00016 Monterotondo Scalo (Roma), Italy Abbreviations: BGE, background electrolyte; HPLC, high performance liquid chromatography; I.S., internal standard; MECC, micellar electrokinetic capillary chromatography; SDS, sodium dodecyl sulrdte
8 VCH Vcrleg?gesellschafi m b H , D-6940 Weinheim,
1992
the detector) S.G.E. (Victoria, Australia) was used for the separations. The modified detector cell and the construction of the capillary connection block, as well as the electrolyte vessel and the capillary holder were described previously [15]. Detection was carried out at 225 nm. Electropherograms were recorded by a Chromatopac C-R5A (Shim a d m , Kyoto, Japan) integrator. Sampling was made by a hydrodynamic method (3 s at a height of 10 cm). The constant voltage mode (10 kV) was chosen for electrophoresis.
2.2 Chemicals Standard compounds of atrazine and simazine were obtained from Supelco I N C (Bellefonte, USA). Sodium dodecyl sulfate (SDS) and hydroquinone monobenzyl ether were obtained from Fluka (Buchs, Switzerland). Methanol (Farmitalia Carlo Erba, Milan, Italy) was HPLC-grade; the water was double distilled. For analysis at pH 7 and 8,lO mM sodium dihydrogen orthophosphate and 25 mM SDS served as BGE, while at pH 9 and 10 it was 10 mM sodium tetraborate and 25 mM SDS. The solutions used for electrophoretic experiments were prepared by dissolving the standard compounds in methanol. Atrazine and simazine standard solutions M - ~ O - ~ M), containing a fixed concentration of hydroquinone monobenzyl ether as internal standard (I.S.), were prepared in B G E and used for calibration. 2.3 Samples Water samples from the river Tiber were used as real samples for calibration graph and quantitative analysis. The extraction procedure was similar to that described in [4]; 200 m L of river water with I m L of 10% NaOH were extracted with 3 x 50 mL portions of chloroform in a separatory funnel. The organic layers were collected, passed through anhydrous sodium sulfate, and dried in a rotating evaporator. The residue was dissolved in 1 mL of BGE, corresponding to a 200 times more concentrated sample. The determination of simazine and atrazine in environmental samples was performed, using the I.S. method, by measuring the peak area ratio.
3 Results and discussion Because of the low p/C, 12 values of atrazine and simazine, either low or high pH values of BGE are necessary for their separation by CZE, which is not convenient for electrophoresis. Alternatively 0173-083S/Y2/0Y 10-0698 $3 S0+.25/0
MECC of atrazine and simazine
ElrcfrophorPsiJ 1992. 13. 698-700
a c
they can be determined as hydroxylated derivatives [I I]. Because of the relatively high stability of these compounds near neutral pH values [16], we decided to perform their separation as uncharged compounds by MECC. SDS was added to the BGE above its critical micellar concentration (CMC) [17] as selective agent. In order to improve the separation, several electrolyte systems, containing 2.5 mM SDS at pH ranges from 7 to 10, were used to study the effect of pH on the capacity factor K of the analyzed substances. The capacity factor, defined as the ratio n,/ndq (n, and n,, represent the total amount of the analyte incorporated into the micelles and the aqueous phase, respectively) were calculated by using formula (1) of [14],
where t,, t,, and to represent the migration time of solute, micelles and methanol, respectively. A 25 mM SDS concentration in BGE was adequate to obtain different apparent electrophoretic mobilities to separate atrazine and simazine. SDS concentrations above 25 mM only increased the retention times without improving the efficiency of separation, whereas in absence of SDS all the compounds migrated at the velocity equal to the electroosmotic flow (methanol) at the selected pH values. Figure 1 shows the pH dependence of the capacity factor K of atrazine and simazine. In the range selected, the influence of pH on the capacity factor of atrazine and simazine seems weak because of their neutrality and low polarity at those pH. The optimum buffer consisted of 10 mM biphosphate buffer, pH 8, and 2.5 mM SDS. This buffer combined complete resolution with short analysis time and good reproducibility of both migration times and substance/I.S. peak area ratios.
-
Capacity Factor k' --------------o(l
6L
-1
699
4
I
1-
0
2
I
I
10
14
min
f'igure2. Electropherogramofthe separation of standard mixtures ofherbicides. For operating conditions see Fig. 1. SIM, simazine; ATR, atrazine; I.S., internal standard.
Table 1. Recoverv on soiking river water samDles Tiber river water sample I 2
3
Simazine wba'
NDb' ND~) 0.99
Atrazine PPb") 0.41 0.45 1.03
a) Values not corrected by the recovery value b) ND, not detected
--
Atrazine
4 -
zine, were carried out and the calibration graph was found to be linear from to M with a correlation coefficient of 0.9974. The relative standard deviation, calculated by measuring the peak area ratio by analyzing the same sample (4 X M herbicides, 3 X 10 M 1,s.)three times, was found to be 1.23%.
Simazine
2-
"
6
7
8
9
10
I1
PH Figure 1. pH effects ofatrazine and simazine on capacity factor K' and internal standard (1,s.). SDS concentration, 25 mM; voltage, 10 kV,20 PA. Detection at 225 nm.
Figure 2 shows a typical electropherogram of the separation of atrazine and simazine under the optimized conditions. The separation efficiency was of the order of lo5 the number of theoretical plates. Ten electrophoretic runs, performed by injecting standard solutions. containing 3 X M hydroquinone monobenzyl ether (I.S.) and different amounts ofatrazine and sima-
The method exhibited a low determination limit of 3.5 X M for both herbicides.Without a preconcentration step, in real samples the determination limit corresponding to 1.75 X M or 0.38 ppb and 0.35 ppb, respectively, for atrazine and simazine, seems feasible. Recoveries were tested in triplicate by spiking samples of river water with three different concentration levels of herbicides, lo-* M,3 X lo-*M, and 5 X M, corresponding, after the preconcentration step, to 2 X M,6 X M and M (Table 1).Quantitative analysis of the concentrated extract of river water samples showed the presence of atrazine while simazine was present below the detection 1imit.The results, corroborated by HPLC analysis [8], are shown in Table 2. In Fig. 3 the electropherogram of a real sample is shown
700
Elecfruphorpsis 1992. 13, 698-700
C. Desiderio and S. Fanali
Table 2. Quantitative results of atrazine and simazine determination in samples oi' river water
Concentration level 1 X 10-8
(2 x 10-6 3 x 10-8 (6 x 10-6 5 X 10-8 (1 x 10-8
Recoveriesa) simazine
Recoveries") atrazine
M
112.0% f 10.2
83.0°/o rt 8.8
M) M
107.3% f 11.1
80.3% k 17.3
117.6% f 16.0
104.3% I2.6
M)h) M
M)b)
a) Mean of three determinations and standard deviation b) Concentration level after preconcentration
4 Concluding remarks Atrazine and simazine can be separated as neutral substances by MECC under optimized conditions, at pH 8, avoiding possible degradation. The method is fast: the separation can be carried out in less than 10 min with good results. The detection limit is about 0.4 ppb for each herbicide, comparable to that obtained by gas chromatography and/or HPLC methods. Further studies are in progress in order to apply MECC to the simultaneous separation of atrazine, simazine and their metabolic products in environmental samples. Received July 28, 1992
5 References
.t cu
= l
si d
1
-y
0
2
6
I
10
14
min
F"gure3. Eleclrophersogram ofrcal sample ofriverwater. 1,atrazine,2,internal standard (IS.). Operating conditions as in Fig. 1. For extraction procedure see Section 2.3.
[l] Berry, J. L., Furm Chemical Handbook, Meister Pubbl., Willonghby, OH 1985. [2] Pelizzetti, E., Maurino, V., Minero, C., Carlin,V., Pramauro. E., Zerbinati, 0. and Tosato, M. L., Environ. Sci. Techno/. 1990, 24, 15591565. [3] Lee, H. B . and Stokker, Y. D., J . Assoc. Ofl Anal. Chem. 1986, 69, 568-572. [4] Baldi, M., Coppi, S., Davi', L., Benedetti, S., Bovolenta, A., Penazzi, L. and Previati, M., Inquinamento 1989, 9, 74-81. [5] Grob, K. and Li, Z., J. Chromatogr. 1989, 473,423-430. [6] Mangani, F. and Bruner, F., Chroinatographia 1989, 17, 377-380. [7] Coquart, V. and Hennion, M. C., J. Chromatogr. 1991, 585, 67-73. [8] Pacacova, V., Stulik, K. and Prihoda, M., J. Chromatogra. 1988,442, 147-1 55. [9] Jork, H. and Roth, B., J . Chromatogr. 1977, 144, 39-56. [lo] Krivankovi, L., BoTek, P., Tekel, J. and Kovacicova, J., ElectrophoreS / S 1989, 10, 731-734. [ l l ] Foret, F., SustaCek,V. and Botek. P., Electrophoresis 1990,II, 95-97. 1121 Terabe, S., Otsuka, K., Ichikawa, K., Tsuchiya, A. and Ando, T., Anal. Chem. 1984, 56, 111-113. [13] Terabe, S., Otsuka, K. and Ando, T., Anal. Chem. 1985.57,834-841. [14] Terabe, S., Trends Anal. Chem. 1989, 8, 129-134. [15] Fanali,S.,Ossicini,L.,Foret,F.and BoEek,P., J. Mwocol.Sep. 1989. I , 190-194. [16] The Merk Index, 11 Edition, Merk, Inc., Rahway, N J 1989, pp. 887, 849 1. [17] Nishi, H. and Terabe, S., Electrophoresis 1990, 11, 691-701.
Ele~crophore.~it 1992.
C . Desiderio and S . Fanali
Claudia Desiderio Salvatore Fanali Istituto di Cromatografia del C.N.R. Monterotondo Scalo, Roma
13,698-700
Atrazine and simazine determination in river water samples by micellar electrokinetic capillary chromatography A rapid and sensitive micellar electrokinetic capillary chromatography analytical method was used for the determination of chlorotriazine herbicides in river water samples. Several electrolyte systems in the pH range 7-10 were tested in order to optimize the separation. The two compounds were separated in less than 10 min and the determination limit was about 0.4 ppb for each herbicide. Kecovery values of the method were in the range 8O0/o-117%.
1 Introduction S-triazine herbicides are among the most widely used pesticides to control broadleaf and grassy weeds in corn and other crops [l]. Due to their extensive use and their relatively high persistence, chlorotriazines contaminate the acquatic environment through agricultural runoff, direct application and leaching into ground water, in concentrations that are increasing [2]. Many efforts have been devoted to develop rapid ascays for the determination of triazine herbicides at low levels in water. Several analytical techniques that have been used so far for the determination of these compounds are gas-chromatography (GC) [3-61, high performance liquid chromatography (HPLC) [2, 5, 7, 81, thin layer chromatography (TLC) [9], isotachophoresis (ITP) [10],and capillary zone electrophoresis (CZE) [ 113.Micellar electrokinetic capillary chromatography (MECC), first introduced by Terabe and co-workers [12, 131, is a relatively new type of separation method, combining electrophoresis and chromatography [14], which is mainly suited for the resolution of neutral compounds. In M E C C an ionic surfactant is added to the background electrolyte (BGE) at a concentration level above its critical micellar concentration. When a high vollage is applied to the system, the uncharged compounds, moving with the same velocity as that of the electroosmotic flow, will separate by different partitioning between the aqueous phase and the micellar one. The last phase moves in the opposite direction to the one before. This paper presents our investigation on the possibility of applying this new electrophoretic technique to the analysis of atrazine and simazine in river water samples.
2 Materials and methods 2.1 Apparatus Electrophoretic experiments were performed in a laboratory-made apparatus consisting of a modified Varian UV 2550 detector and a power supply (Glassman LH60R) able to deliver either constant voltage or constant current. A fused silica capillary tube (50 cm long X 75 pm ID, 36 cm to
Correspondence: Dr. S. Fanali,Istituto di Cromatografia del C.N.R.,Area della Ricerca di Roma, P.O. Box 10,I-00016 Monterotondo Scalo (Roma), Italy Abbreviations: BGE, background electrolyte; HPLC, high performance liquid chromatography; I.S., internal standard; MECC, micellar electrokinetic capillary chromatography; SDS, sodium dodecyl sulrdte
8 VCH Vcrleg?gesellschafi m b H , D-6940 Weinheim,
1992
the detector) S.G.E. (Victoria, Australia) was used for the separations. The modified detector cell and the construction of the capillary connection block, as well as the electrolyte vessel and the capillary holder were described previously [15]. Detection was carried out at 225 nm. Electropherograms were recorded by a Chromatopac C-R5A (Shim a d m , Kyoto, Japan) integrator. Sampling was made by a hydrodynamic method (3 s at a height of 10 cm). The constant voltage mode (10 kV) was chosen for electrophoresis.
2.2 Chemicals Standard compounds of atrazine and simazine were obtained from Supelco I N C (Bellefonte, USA). Sodium dodecyl sulfate (SDS) and hydroquinone monobenzyl ether were obtained from Fluka (Buchs, Switzerland). Methanol (Farmitalia Carlo Erba, Milan, Italy) was HPLC-grade; the water was double distilled. For analysis at pH 7 and 8,lO mM sodium dihydrogen orthophosphate and 25 mM SDS served as BGE, while at pH 9 and 10 it was 10 mM sodium tetraborate and 25 mM SDS. The solutions used for electrophoretic experiments were prepared by dissolving the standard compounds in methanol. Atrazine and simazine standard solutions M - ~ O - ~ M), containing a fixed concentration of hydroquinone monobenzyl ether as internal standard (I.S.), were prepared in B G E and used for calibration. 2.3 Samples Water samples from the river Tiber were used as real samples for calibration graph and quantitative analysis. The extraction procedure was similar to that described in [4]; 200 m L of river water with I m L of 10% NaOH were extracted with 3 x 50 mL portions of chloroform in a separatory funnel. The organic layers were collected, passed through anhydrous sodium sulfate, and dried in a rotating evaporator. The residue was dissolved in 1 mL of BGE, corresponding to a 200 times more concentrated sample. The determination of simazine and atrazine in environmental samples was performed, using the I.S. method, by measuring the peak area ratio.
3 Results and discussion Because of the low p/C, 12 values of atrazine and simazine, either low or high pH values of BGE are necessary for their separation by CZE, which is not convenient for electrophoresis. Alternatively 0173-083S/Y2/0Y 10-0698 $3 S0+.25/0
MECC of atrazine and simazine
ElrcfrophorPsiJ 1992. 13. 698-700
a c
they can be determined as hydroxylated derivatives [I I]. Because of the relatively high stability of these compounds near neutral pH values [16], we decided to perform their separation as uncharged compounds by MECC. SDS was added to the BGE above its critical micellar concentration (CMC) [17] as selective agent. In order to improve the separation, several electrolyte systems, containing 2.5 mM SDS at pH ranges from 7 to 10, were used to study the effect of pH on the capacity factor K of the analyzed substances. The capacity factor, defined as the ratio n,/ndq (n, and n,, represent the total amount of the analyte incorporated into the micelles and the aqueous phase, respectively) were calculated by using formula (1) of [14],
where t,, t,, and to represent the migration time of solute, micelles and methanol, respectively. A 25 mM SDS concentration in BGE was adequate to obtain different apparent electrophoretic mobilities to separate atrazine and simazine. SDS concentrations above 25 mM only increased the retention times without improving the efficiency of separation, whereas in absence of SDS all the compounds migrated at the velocity equal to the electroosmotic flow (methanol) at the selected pH values. Figure 1 shows the pH dependence of the capacity factor K of atrazine and simazine. In the range selected, the influence of pH on the capacity factor of atrazine and simazine seems weak because of their neutrality and low polarity at those pH. The optimum buffer consisted of 10 mM biphosphate buffer, pH 8, and 2.5 mM SDS. This buffer combined complete resolution with short analysis time and good reproducibility of both migration times and substance/I.S. peak area ratios.
-
Capacity Factor k' --------------o(l
6L
-1
699
4
I
1-
0
2
I
I
10
14
min
f'igure2. Electropherogramofthe separation of standard mixtures ofherbicides. For operating conditions see Fig. 1. SIM, simazine; ATR, atrazine; I.S., internal standard.
Table 1. Recoverv on soiking river water samDles Tiber river water sample I 2
3
Simazine wba'
NDb' ND~) 0.99
Atrazine PPb") 0.41 0.45 1.03
a) Values not corrected by the recovery value b) ND, not detected
--
Atrazine
4 -
zine, were carried out and the calibration graph was found to be linear from to M with a correlation coefficient of 0.9974. The relative standard deviation, calculated by measuring the peak area ratio by analyzing the same sample (4 X M herbicides, 3 X 10 M 1,s.)three times, was found to be 1.23%.
Simazine
2-
"
6
7
8
9
10
I1
PH Figure 1. pH effects ofatrazine and simazine on capacity factor K' and internal standard (1,s.). SDS concentration, 25 mM; voltage, 10 kV,20 PA. Detection at 225 nm.
Figure 2 shows a typical electropherogram of the separation of atrazine and simazine under the optimized conditions. The separation efficiency was of the order of lo5 the number of theoretical plates. Ten electrophoretic runs, performed by injecting standard solutions. containing 3 X M hydroquinone monobenzyl ether (I.S.) and different amounts ofatrazine and sima-
The method exhibited a low determination limit of 3.5 X M for both herbicides.Without a preconcentration step, in real samples the determination limit corresponding to 1.75 X M or 0.38 ppb and 0.35 ppb, respectively, for atrazine and simazine, seems feasible. Recoveries were tested in triplicate by spiking samples of river water with three different concentration levels of herbicides, lo-* M,3 X lo-*M, and 5 X M, corresponding, after the preconcentration step, to 2 X M,6 X M and M (Table 1).Quantitative analysis of the concentrated extract of river water samples showed the presence of atrazine while simazine was present below the detection 1imit.The results, corroborated by HPLC analysis [8], are shown in Table 2. In Fig. 3 the electropherogram of a real sample is shown
700
Elecfruphorpsis 1992. 13, 698-700
C. Desiderio and S. Fanali
Table 2. Quantitative results of atrazine and simazine determination in samples oi' river water
Concentration level 1 X 10-8
(2 x 10-6 3 x 10-8 (6 x 10-6 5 X 10-8 (1 x 10-8
Recoveriesa) simazine
Recoveries") atrazine
M
112.0% f 10.2
83.0°/o rt 8.8
M) M
107.3% f 11.1
80.3% k 17.3
117.6% f 16.0
104.3% I2.6
M)h) M
M)b)
a) Mean of three determinations and standard deviation b) Concentration level after preconcentration
4 Concluding remarks Atrazine and simazine can be separated as neutral substances by MECC under optimized conditions, at pH 8, avoiding possible degradation. The method is fast: the separation can be carried out in less than 10 min with good results. The detection limit is about 0.4 ppb for each herbicide, comparable to that obtained by gas chromatography and/or HPLC methods. Further studies are in progress in order to apply MECC to the simultaneous separation of atrazine, simazine and their metabolic products in environmental samples. Received July 28, 1992
5 References
.t cu
= l
si d
1
-y
0
2
6
I
10
14
min
F"gure3. Eleclrophersogram ofrcal sample ofriverwater. 1,atrazine,2,internal standard (IS.). Operating conditions as in Fig. 1. For extraction procedure see Section 2.3.
[l] Berry, J. L., Furm Chemical Handbook, Meister Pubbl., Willonghby, OH 1985. [2] Pelizzetti, E., Maurino, V., Minero, C., Carlin,V., Pramauro. E., Zerbinati, 0. and Tosato, M. L., Environ. Sci. Techno/. 1990, 24, 15591565. [3] Lee, H. B . and Stokker, Y. D., J . Assoc. Ofl Anal. Chem. 1986, 69, 568-572. [4] Baldi, M., Coppi, S., Davi', L., Benedetti, S., Bovolenta, A., Penazzi, L. and Previati, M., Inquinamento 1989, 9, 74-81. [5] Grob, K. and Li, Z., J. Chromatogr. 1989, 473,423-430. [6] Mangani, F. and Bruner, F., Chroinatographia 1989, 17, 377-380. [7] Coquart, V. and Hennion, M. C., J. Chromatogr. 1991, 585, 67-73. [8] Pacacova, V., Stulik, K. and Prihoda, M., J. Chromatogra. 1988,442, 147-1 55. [9] Jork, H. and Roth, B., J . Chromatogr. 1977, 144, 39-56. [lo] Krivankovi, L., BoTek, P., Tekel, J. and Kovacicova, J., ElectrophoreS / S 1989, 10, 731-734. [ l l ] Foret, F., SustaCek,V. and Botek. P., Electrophoresis 1990,II, 95-97. 1121 Terabe, S., Otsuka, K., Ichikawa, K., Tsuchiya, A. and Ando, T., Anal. Chem. 1984, 56, 111-113. [13] Terabe, S., Otsuka, K. and Ando, T., Anal. Chem. 1985.57,834-841. [14] Terabe, S., Trends Anal. Chem. 1989, 8, 129-134. [15] Fanali,S.,Ossicini,L.,Foret,F.and BoEek,P., J. Mwocol.Sep. 1989. I , 190-194. [16] The Merk Index, 11 Edition, Merk, Inc., Rahway, N J 1989, pp. 887, 849 1. [17] Nishi, H. and Terabe, S., Electrophoresis 1990, 11, 691-701.
