Cloud Point Extraction as a Preconcentration Step Prior to Capillary

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Anal. Chem. 1999, 71, 2468-2474

Cloud Point Extraction as a Preconcentration Step Prior to Capillary Electrophoresis R. Carabias-Martı´nez,* E. Rodrı´guez-Gonzalo, J. Domı´nguez-Alvarez, and J. Herna´ndez-Me´ndez

Departamento de Quı´mica Analı´tica, Nutricio´ n y Bromatologı´a, Facultad de Quı´mica, Universidad de Salamanca, 37008 Salamanca, Spain

Cloud point extraction was applied as a preconcentration step prior to capillary electrophoresis. The behavior of a surfactant-rich micellar phase injected into a capillary electrophoresis system was studied using different separation modes: micellar electrokinetic capillary chromatography and capillary zone electrophoresis (CZE). A problem that appeared on introducing a surfactant-rich phase into a bare fused silica capillary was that the surfactant was adsorbed onto the wall of the capillary, leading to a marked loss of efficiency and reproducibility both in the migration times and in the areas of the electrophoretic peaks. The use of cetyltrimethylammonium bromide dynamically coated capillaries afforded reproducible results, although the half-life of the capillary was short. The most satisfactory results were obtained by using nonaqueous media in the CZE mode, thus avoiding surfactant adsorption. Other parameters related to the composition of the injection medium were also studied to optimize the electrophoretic behavior of the analytes and the sensitivity of the determination. The optimized procedure was applied to the determination of triazines in tap and river water samples. Capillary electrophoresis (CE) is an especially useful technique in both biochemical and pharmaceutical analysis, although in recent years its applicability in other fields of chemical analysis has also been demonstrated.1 However, the major drawback in CE is low system loadability. This has aroused considerable interest both in the development of improvements in the sensitivity of detection systems and in the study of different preconcentration techniques for the determination of trace-level amounts. In recent years, the applications of CE to the determination of pollutants in samples of environmental interest have increased considerably2-7 although the field continues to be underdeveloped. The use of preconcentration steps based on phase separation by cloud point extraction (CPE)8-10 offers a convenient alternative * Corresponding author: (fax) (+) 923 29 45 74; (e-mail) [email protected]. (1) Kurh, N. G.; Monning, C. A. Anal. Chem. 1992, 64, 389R. (2) Bruin, G. J. M.; Tock, P. P. H.; Kraak, J. C.; Poppe, H. J. Chromatogr. 1990, 517, 557. (3) Ong, C. P.; Ng, C. P.; Lee, H. K.; Li, S. F. Y. J. Chromatogr. 1991, 542, 473. (4) Nielen, M. W. F. J. Chromatogr. 1991, 637, 81. (5) Cai, J.; El-Rassi, Z. J. Liq. Chromatogr. 1992, 15, 1193. (6) Dinelli, G.; Vicari, A.; Catizone, P. J. Agric. Food Chem. 1993, 41, 742. (7) Gaitonde, C. D.; Pathak, P. J. Chromatogr. 1990, 514, 389. (8) Watanabe H. In Solution Behaviour of Surfactants; Mittal, K. L., Fendler, E. F., Eds.; Plenum Press: New York, 1992; Vol. 2, pp 1305-1313.

2468 Analytical Chemistry, Vol. 71, No. 13, July 1, 1999

to more conventional extraction systems. The small volume of the surfactant-rich phase obtained with this methodology permits the design of extraction schemes that are simple, cheap, and of lower toxicity than extractions that use organic solvents and also provides results similar to those obtained with other separation techniques. It can be used to separate and/or preconcentrate analytes as a step prior to their determination in hydrodynamic analytical systems such as high-perfomance liquid chromatography (HPLC)11-13 or flow injection analysis (FIA).14 However, its application as a preconcentration step prior to analysis by capillary electrophoresis has not yet been described. Triazine herbicides have been used as test compounds because their electrophoretic behavior is already known. These herbicides are widely used as selective pre- and postemergence herbicides for the control of broadleaf and grassy weeds in many agricultural crops. They are basic compounds15 that protonate in an acidic medium to yield a cationic form (HA+). Several procedures employing capillary zone electrophoresis (CZE) have been proposed16,17 for the determination of methylthiotriazines. However, chlorotriazines (pKa values of about 1.6 in aqueous medium) are mainly separated as neutral species by micellar electrokinetic capillary chromatography (MECC)18-21 because these species require a strongly acidic medium to be converted into the protonated cationic form. Recently, the simultaneous separation of chloro- and methylthiotriazines by CZE in nonaqueous media has been described.22 (9) Moreno Cordero, B.; Pe´rez Pavo´n, J. L.; Garcı´a Pinto, C.; Ferna´ndez Laespada, M. E. Talanta 1993, 40, 1703-1710. (10) Hinze, W. L.; Pramauro, E. CRC Crit. Rev. Anal. Chem. 1993, 24, 133177. (11) Saitoh, T.; Hinze, W. L. Anal. Chem. 1991, 63, 2520-2525. (12) Garcı´a Pinto, C.; Pe´rez Pavo´n, J. L.; Moreno Cordero, B. Anal. Chem. 1995, 67, 2606-2612. (13) Carabias Martı´nez, R.; Rodrı´guez Gonzalo, E.; Garcia Jime´nez, M. G.; Garcı´a Pinto, C.; Pe´rez Pavo´n, J. L.; Herna´ndez Me´ndez, J. J. Chromatogr., A 1996, 754, 85-96. (14) Ferna´ndez Laespada, M. E.; Pe´rez Pavo´n, J. L.; Moreno Cordero, B. Analyst (Cambridge, U.K.) 1993, 118, 209-2212. (15) Weber, J. B. Residue Rev. 1970, 32, 93-130. (16) Foret, F.; Sustacek, V.; Bocek, P. Electrophoresis 1990, 11, 95-97. (17) Cai, J.; El-Rassi, Z. J. Liq. Chromatogr. 1992, 15, 1179-1192. (18) Cai, J.; El-Rassi, Z. J. Chromatogr. 1992, 608, 31. (19) Desiderio, C.; Fanalli, S. Electrophoresis 1993, 13, 698. (20) Dinelli, G.; Bonetti, A.; Catizone, P.; Galletti, G. C. J. Chromatogr., B 1994, 656, 275. (21) Carabias Martı´nez, R.; Rodrı´guez Gonzalo, E.; Mun ˜oz Domı´nguez, A. I.; Domı´nguez Alvarez, J.; Herna´ndez Me´ndez, J. J. Chromatogr., A 1996, 733, 349-360. (22) Carabias-Martı´nez, R.; Rodrı´guez-Gonzalo, E.; Domı´nguez-Alvarez, J.; Herna´ndez-Me´ndez, J. Anal. Chem. 1997, 69, 4437-4444. 10.1021/ac9812227 CCC: $18.00

© 1999 American Chemical Society Published on Web 05/14/1999

This work evaluates CPE as a preconcentration step prior to analysis by capillary electrophoresis. Since the cloud point preconcentration methodology has already been reported, our work focused on the particular problems involved in its application in a capillary electrophoresis system; specifically, the search for electrophoretic conditions in which the surfactant will not be adsorbed onto the capillary walls. In this paper, it is shown that the use of nonaqueous media permits satisfactory separation of samples with high contents in nonionic surfactants. To optimize the sensitivity of analyte determination, a detailed study was carried out on the composition that the injected sample should have: the electrophoretic separation medium used was 50:50 (v/ v) acetonitrile-methanol, 10 mM perchloric acid, and 20 mM sodium dodecyl sulfate (SDS). The proposed method was used for the determination of triazines in natural water samples such as drinking and river water. EXPERIMENTAL SECTION Apparatus. Capillary electrophoresis was performed with a P/ACE 2000 (Beckman Instruments, CA) apparatus equipped with a UV detector. Standard P/ACE capillaries were used: 75 µm internal diameter, 57 cm long, detection at 50 cm. A Kokusan H-103 N centrifuge was used for the separation of the two phases obtained by the cloud point methodology. A rotovapor (Buchi, Switzerland) was used for evaporating off the water in the surfactant-rich phase. Reagents. All triazine herbicides were obtained from Riedel de Hae¨n (Seelze-Hannover, Germany) and were used without further purification (minimum percent purity greater than 98%). The chlorotriazines studied were as follows: Atrazine, 2-chloro4-(ethylamino)-6-(isopropylamino)-1,3,5-triazine; Simazine, 2-chloro4,6-bis(ethylamino)-1,3,5-triazine; and Propazine, 2-chloro-4,6-bis(isopropylamino)-1,3,5-triazine. The methylthiotriazines studied were Ametryne, 2-(methylthio)-4-(ethylamino)-6-(isopropylamino)1,3,5-triazine; Prometryne, 2-(methylthio)-4,6-bis(isopropylamino)1,3,5-triazine; and Terbutryne, 2-(methylthio)-4-(ethylamino)-6(terbutylamino)-1,3,5-triazine. Stock solutions of each triazine were prepared in methanol at 500 µg/mL. Sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), and the nonionic surfactant Triton X-114 were obtained from Fluka (Buchs, Switzerland) and were used without further purification. The organic solvents, acetonitrile (AN) and methanol (MeOH), were of HPLC grade (Carlo Erba, Milan, Italy) and were used as received. Ultrahigh-quality water obtained with an Elgastat UHQ water-purification system was used. All chemicals used for the preparation of the buffer electrolytes were of analytical reagent grade. Procedure. Cloud Point Preconcentration. One-fourth gram of Triton X-114 was added to a 100-mL volume of an aqueous solution containing the six triazines; a 10-mL aliquot of this was placed in a centrifuge tube and heated for 10 min in a thermostated bath at 40 °C, triggering the cloud point effect and the appearance of two phases. These two phases were separated by centrifugation for 15 min at 3500 rpm (2195g). The centrifuge tube with both phases formed was kept in a freezer for 10-15 min. Then, the surfactant-rich phase was isolated by pouring off the aqueous solution in the upper part. CZE Separation of Triazines in Nonaqueous Medium. Uncoated capillaries were used throughout the study. Before use,

all new capillaries were pretreated as described elsewhere.22 Before each injection, the capillary was rinsed for 2 min with methanol and 3 min with the separation buffer, applying a potential of 22 kV. Electrokinetic injection at 5 kV over 15 s was used to introduce the samples into the capillary. Analyses were performed with an applied voltage of 22 kV with the capillary thermostated at 25 ( 1 °C and with UV detection at 214 nm. The separation medium was 50:50 (v/v) acetonitrile-methanol, 10 mM perchloric acid, and 20 mM SDS. Electrolyte solutions of perchloric acid in 50:50 (v/v) acetonitrile-methanol were freshly prepared and not used after long storage since SDS run buffers may undergo acidcatalyzed hydrolysis. To obtain the sample for injection, a 100-µL aliquot of the surfactant-rich phase was collected with a Hamilton syringe and placed in the rotovapor at 70 °C for 10 min to remove the water. The dry residue thus obtained was diluted with 100 µL of a solution containing 70:30 (v/v) acetonitrile-methanol and 2 mM perchloric acid. Safety considerations: Nonaqueous Perchloric acid solutions containing N-compounds should never be allowed to concentrate, dry, or be heated because this would yield perchlorate salts of N-compounds, which are typically dangerous and may explode. Analysis of Triazine Herbicides in Water Samples. Samples of river water were taken from the River Tormes in the city of Salamanca (Spain). They were collected directly in 1-L glass containers, stored at 4 °C in the dark, and analyzed within 24 h after collection. All river-water samples were filtered through sintered glass filters (no. 5) to remove suspended particulate matter before use. Deionized and tap water samples were used without any further treatment. Spiked water samples were prepared by placing 0.5-2.5 mL of the triazine mixture in methanol, at the appropriate concentration, in a volumetric flask and filling to 100 mL with the water sample. Also, 0.25 g of Triton X-114 was added. Ten-milliliter aliquots of this solution were taken to accomplish cloud point preconcentration. Nonspiked samples were analyzed following the same procedure to check for the presence of the herbicides under study. For analyte quantification, the normalized peak areas (NPA) were processed by dividing the observed peak area values by their corresponding migration times. In the determination of triazines in natural water samples, the internal standard method was used. Ametryne was chosen as the internal standard for terbutryne and prometryne, while propazine was used as the internal standard for simazine and atrazine. In turn, terbutryne served as the internal standard for ametryne, and atrazine served as the internal standard for propazine. The values of the normalized peak areas with the internal standard (NPAis ) NPAtriazine/NPAinternal standard) were used in the quantification of the river and tap water samples. RESULTS AND DISCUSSION MECC Separation of Triazines in the Presence of SDS and CTAB. Micellar electrokinetic capillary chromatography (MECC) for the simultaneous separation of chloro- and methylthiotriazines has been reported previously,21 using 60 mM borate buffer, pH 9.2, and 50 mM SDS as the separation medium. The experiments conducted using this separation medium showed that consecutive injections of a phase rich in the surfactant Triton X-114 produced a continuous increase in the migration times of the triazines. This Analytical Chemistry, Vol. 71, No. 13, July 1, 1999

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is due to the adsorption of Triton X-114 onto the walls of the capillary, the performance of which deteriorates progressively. It was observed experimentally that it was not possible to regenerate the initial conditions of the capillary wall with any of the washing steps assayed. Neither was it possible to achieve a steady state in which increases in the migration times of the analytes ceased to progress. The adsorption of Triton X-114 onto the capillary wall can be controlled using dynamic coatings, such as those obtained when the cationic surfactant CTAB is added to the separation buffer. In this case, the run buffer used was 20 mM phosphate buffer, pH 8.0, 60 mM CTAB, 10% (v/v) methanol, 10% (v/v) acetonitrile. However, it should be noted that although this solution is fairly simple, the half-life of capillaries coated with CTAB and then used in the separation of Triton X-114-rich phases is relatively short. To date, no procedure that restores the initial behavior of the capillary has been found; it is therefore necessary to use a new capillary after the previous one has deteriorated. A detailed description of MECC separation of triazines after CPE is included as Supporting Information. CZE Separation of Triazines in Nonaqueous Medium. Triazines are basic species able to become protonated in acidic media. In an aqueous medium, the pKa values of chlorotriazines are about 1.6, whereas those of methylthiotriazines are close to 4. In an aqueous acidic medium, separation by CZE of chlorotriazines21 is difficult owing to the low pH values required for their conversion into protonated cationic form (HA+). However, the use of nonaqueous media22 modifies the acidbase characteristics of these analytes, permitting their separation by CZE. The possibility of injecting a surfactant-rich sample in a nonaqueous electrophoretic medium to overcome adsorption of the surfactant onto the capillary walls is very attractive. To attempt this, surfactant-rich samples were suitably diluted and injected in a nonaqueous separation medium, 10 mM perchloric acid in 50:50 (v/v) acetonitrile-methanol and 20 mM SDS. Under these conditions, satisfactory separation was obtained for a mixture of chloro- and methylthiotriazines in a short analysis time. Repeated injection of a surfactant-rich sample in this nonaqueous medium afforded reproducible electropherograms, with no modifications in the migration times, together with suitable resolution among the electrophoretic peaks of the six triazines assayed. Apart from the signal corresponding to the triazines, two new signals appeared; these were due to the migration of the surfactant but did not interfere with the peaks of the analytes (Figure 1). Optimization of the Composition of the Sample Injected. On performing preconcentrations using CPE, the sensitivity of the preconcentration procedure is a function of the ratio of the initial volume of sample to be preconcentrated to the volume of surfactant-rich phase obtained. The volume of the surfactant-rich phase obtained following phase separation depends on the percentage of surfactant and, also, on the initial volume to be preconcentrated. Additionally, it is necessary to work under conditions in which the volume of the surfactant-rich phase is easy to handle (that is, at least 200-250 µL). This surfactant-rich phase contains preconcentrated analytes together with a certain amount of water and surfactant. On working with a 10.0 mL sample volume and a 2470 Analytical Chemistry, Vol. 71, No. 13, July 1, 1999

Figure 1. CZE electropherogram of a surfactant-rich phase. Separation electrolyte, 10 mM perchloric acid in 50:50 (v/v) acetonitrile-methanol, 20 mM SDS. Electrokinetic injection, at 5 kV over 15 s. Applied voltage, 22 kV. Peaks: (Sz) simazine, (Az) atrazine, (Am) ametryne, (Pz) propazine, (Pm) prometryne, (Tb) terbutryne, (Tx) nonionic surfactant Triton X-114.

percentage of surfactant of 0.25% (w/v), the surfactant-rich phase obtained is 250 µL and contains 91% (v/v) of water and 9% (v/v) of Triton X-114.23 The surfactant-rich phase, as obtained after phase separation, is very viscous. Accordingly, it cannot be injected directly into the electrophoretic system and must be suitably diluted beforehand. Initially, a 2 mM perchloric acid solution in acetonitrile was used for the dilution: the perchloric acid is required to place the triazines in the appropriately acidic medium for their electrokinetic injection. Optimization of the method requires a detailed study of the composition of the injection medium: the percentage of water, the percentage of nonionic surfactant, the presence of organic solvents, and the concentration of perchloric acid. All studies were carried out using 10 mM perchloric acid in 50:50 (v/v) acetonitrile-methanol medium as separation buffer. Effect of the Water Present in the Injected Sample. As indicated above, the surfactant-rich phase is formed of water and the nonionic surfactant Triton X-114. As the presence of water may affect the amount of triazine injected electrokinetically, several samples with 5 ppm of each triazine were prepared in acetonitrile with a concentration of perchloric acid of 2 mM, keeping the percentage of Triton at 10% (w/v) and modifying the percentage of water between 0 and 10% (v/v). The electropherograms thus obtained did not reveal any variations in the migration times of the triazines. However, the normalized peak area of the chlorotriazines (represented by simazine) underwent a decrease when the percentage of water was increased, whereas that of the methylthiotriazines (represented by ametryne) only varied slightly (Figure 2). This kind of behavior can be explained in terms of the acidbase characteristics of both families of triazines. As previously reported,22 in a 50:50 (v/v) acetonitrile-methanol medium, the pKa* values of chlorotriazines are close to 3 while those of methylthiotriazines are close to 6. For both families of triazines, these pKa* values are higher than the pKa values observed in an aqueous medium (1.6 and 4 for the chloro- and methylthiotriazines, respectively). (23) Domı´nguez-Alvarez, J., personal communication.

Figure 2. Variation in normalized peak area (NPA) of chlorotriazines (represented only by simazine) and methylthiotriazines (represented only by ametryne) as a function of the percentage of water present in the injected sample.

On carrying out electrokinetic injection, the amount of analyte injected depends on its electrophoretic mobility and hence on the charged fraction of analyte. This could explain why the normalized area of the chlorotriazines decreases considerably when the water content of the sample injected is increased. When the water content is increased, the pKa* of the chlorotriazines would be expected to decrease and hence also the charged fraction present in them. To obtain maximum sensitivity for the chlorotriazines, the water present in the surfactant-rich phase was eliminated. It was observed that, by carrying out evaporation in a rotovapor at temperatures between 70 and 90 °C over 10 min, the analytical signals of the triazines remained constant. In later studies the water was therefore evaporated off at 70 °C for 10 min. Effect of Percentage of Triton X-114 in the Injected Sample. With a view to determining the effect of Triton X-114 on the analytical signals, a study was carried out modifying the concentration of surfactant in the sample injected and keeping the concentration of triazines constant. Figure 3 shows the variations in the normalized peak areas (NPA) as a function of the percentage of Triton X-114. It may be seen that the normalized peak area is a function of the percentage of surfactant present in the sample injected. It should be noted that successive injections of surfactant-rich samples did not lead to any variations in the migration times of the triazines. Dilution of the surfactant-rich phase prior to injection in the electrophoretic system leads simultaneously to a decrease of the surfactant in the sample and a decrease in the concentration of triazines present in it. These two effects are opposite since the first one has a positive effect on the analytical signal (Figure 3a), while dilution of the analytes should elicit a decrease in the signal. The overall effects of these two opposite phenomena are shown in Figure 3b, which depicts the normalized peak area obtained when a single sample was progressively diluted. The zone of maximum sensitivity is seen to appear for a final Triton X-114 concentration of 15% (w/v). Despite this, it is appropriate to work with a slightly lower final Triton X-114 concentration, 10% (w/v), because frequent drops in the current occur when the sample contains higher surfactant concentrations.

Figure 3. Effect of percentage of Triton X-114 present in the injected sample on normalized peak area (NPA) for ametryne (O) and simazine (0). (a) Variation in NPA, with a constant concentration of triazine, as the concentration of Triton X-114 is modified; (b) variation in NPA upon dilution of a single sample containing 5.0 ppm of triazines and 20% (w/v) of Triton X-114.

Effect of the Percentage of Methanol in the Sample Injected. Although in previous studies dilution had been carried out with a solution of 2 mM perchloric acid in pure acetonitrile, it was observed that the use of methanol-acetonitrile mixtures provided the best results, with a positive effect on the intensity of the analytical signals. Figure 4 shows the values of the normalized peak areas as a function of the percentage of methanol in the injection sample for a series of samples with amounts of methanolacetonitrile varying between 6 and 100% (v/v). The figure also shows the variation in viscosity (η) of the methanol-acetonitrile mixtures.24 Since injection was electrokinetic, the amount of sample injected (Q) is a function of the following parameters:

Q ) (µep + µeo)πr2ECt

(1)

Here, µep is the electrophoretic mobility of the analyte, µeo is the (24) Janz, G. J.; Tomkins, R. P. T. Nonaqueous Electrolytes Handbook, Vol. I; Academic Press: New York, 1973.

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Figure 4. Effect of percentage of methanol present in the sample injected on normalized peak area (NPA) for ametryne (O) and simazine (0). Symbol (b) represents variation in viscosity of methanolacetonitrile mixtures. Sample injected: 5 ppm of each triazine, 2 mM perchloric acid, 10% (w/v) Triton X-114, and varying proportions of methanol in acetonitrile.

electroosmotic mobility, r is the internal radius of the capillary, E is the electric field applied, C is the concentration of the analytes, and t is the injection time. In this case µep . µeo, and also, µep is inversely proportional to the viscosity (η) of the medium. Accordingly, eq 1 can be rewritten as:

Q ≈ µepπr2ECt ) f(1/η)

(2)

It would therefore be expected that the injected amount would increase with the decrease in the viscosity of the sample to be injected. Figure 4 also shows that maximum sensitivity is, indeed, obtained for methanol percentages ranging between 20 and 30% (v/v), values for which the viscosity of the methanolacetonitrile mixtures is minimum. In later studies, the surfactantrich sample was diluted with a 70:30 (v/v) acetonitrile-methanol solution. Effect of Concentration of Perchloric Acid in the Injection Sample. Addition of perchloric acid (HClO4) to the injection sample comes from the need to provide a suitable acidic medium for electrokinetic injection of the triazines in protonated cationic form (HA+). Figure 5a shows the variations in the normalized peak areas on modifying the perchloric acid concentration, between 2 and 13 mM, in the injection sample. The behavior of the chlorotriazines differed from that of the methylthiotriazines, the latter showing a more marked decrease in area than the chlorotriazines. The highest sensitivity was obtained when the sample contained a 2 mM concentration of perchloric acid. On increasing the concentration of perchloric acid in the sample, the charged fraction of the chlorotriazines increased, while that of the methylthiotriazines remained constant. Considering this phenomenon, alone, the signal of the chlorotriazines should increase and that of the methylthiotriazines should remain constant. However, it is also necessary to take into account that, on increasing the concentration of perchloric acid, the salt 2472 Analytical Chemistry, Vol. 71, No. 13, July 1, 1999

Figure 5. Influence of concentration of perchloric acid present in the sample injected. (a) Variation in normalized peak area (NPA) for ametryne (O) and simazine (0); (b) relationship between NPA for ametryne and simazine and the reciprocal of the concentration of perchloric acid. Sample injected: 5 ppm of each triazine, 10% (w/v) Triton X-114, 70:30 (v/v) acetonitrile-methanol, and varying concentrations of perchloric acid.

concentration of the injection medium also increases, generating a decrease in the electric field (E) in this zone because E ) f (C-1HClO4). This phenomenon would affect both families of triazines equally and, considered alone, would lead to a decrease of the same kind in the signals of all the triazines. Nevertheless, the joint action of both phenomena produces a different variation in the normalized peak areas, depending on whether the triazines are chloro- or methylthio-substituted. The normalized peak area (NPA) of an analyte is

NPA ) BµepE ) Bµep+

(

)

1 E 1 + (Ka*/CHClO4)

(3)

where the term B includes all the parameters that are held constant during the experiments, µep+ is the electrophoretic mobility of the cationic protonated form (HA+) of the triazine, Ka* is the nonaqueous acid-base constant, and CHClO4 is the concentration of perchloric acid in the injection sample.

Table 1. NPA Ratio (NPAmethylthiotriazine/NPAchlorotriazine) vs C-1HClO4 Regression Lines and Calculated Values of pKa* for the Chlorotriazines in 70:30 (v/v) Acetonitrile-Methanol NPA ratio vs C-1HClO4 (L mol-1) NPA ratio simazine Am/Sz Tb/Sz Pm/Sz atrazine Am/Az Tb/Az Pm/Az propazine Am/Pz Tb/Pz Pm/Pz

slope × 10-3

intercept

r2

pKa* a

2.366 2.136 2.343

0.944 0.897 0.836

0.977 0.981 0.989

2.6 2.6 2.6

2.008 1.810 1.995

0.890 0.845 0.790

0.941 0.949 0.958

2.7 2.7 2.6

1.296 1.143 1.338

1.109 1.052 0.990

0.831 0.823 0.881

2.9 3.0 2.9

pKa* b

pKac 1.65

1.68

3.2

1.85

a pK * values in 70:30 (v/v) acetonitrile-methanol, calculated from a these regression data. b pKa* values in 50:50 (v/v) acetonitrilemethanol from ref 22. c pKa data in aqueous medium from ref 15.

For the case of a methylthiotriazine (with symbol S) it can be assumed that CHClO4 . Ka*, such that µepS ≈ µepS+, where µepS+ is the electrophoretic mobility of the protonated cationic form of the methylthiotriazine. In the case of a chlorotriazine (with symbol R) it may be assumed that the magnitude of CHClO4 is comparable to the acidbase constant, in this case represented by K /a,R. On relating the normalized peak area of any methylthiotriazine and the normalized peak area of the chlorotriazine considered, it is possible to isolate the effect of the charged fraction of the chlorotriazine since the effect of the electric field is common to both:

(

)

K /a,R NPAS BS µepS+ ) 1+ NPAR BR µepR+ CHClO4

(4)

NPAS BS µepS+ BS µepS+ / 1 ) + K NPAR BR µepR+ BR µepR+ a,R CHClO4

(5)

It was observed experimentally that the plot of this quotient of normalized peak areas against the reciprocal of the concentration of perchloric acid is a linear function (Figure 5b, Table 1).

Additionally, the slope/intercept ratio affords a value that must approach the K /a,R of the chlorotriazines in the organic medium. Table 1 shows the pK /a,R values obtained for the chlorotriazines in the injection medium, 70:30 (v/v) acetonitrile-methanol, comparing them with those reported in an aqueous medium.15 It should be stressed that the value obtained for propazine in this medium (pK /a,R ) 2.9 as the mean value) is close to that calculated previously (pKa* ) 3.2) in 50:50 (v/v) methanolacetonitrile medium.22 Analytical Data. Optimization of the variables affecting the sample to be injected can be summarized as follows: (1) The amount of water present must be as low as possible and hence its evaporation in a rotovapor is recommended. (2) The percentage of acetonitrile-methanol in the sample should be 70:30 (v/v). (3) The concentration of perchloric acid should be close to 0.002 mol L-1 in order to obtain maximum sensitivity for these analytes. (4) The final percentage of Triton X-114 should be close to 10% (w/ v). The extraction step by CPE is carried out using a 10.0 mL sample volume and a percentage of Triton X-114 of 0.25% (w/v); thus, the surfactant-rich phase obtained is 250 mL and contains 91% (v/v) of water and 9% (v/v) of Triton X-114. A 100-µL aliquot of this surfactant-rich phase is taken, and after the water has been evaporated off, the dry residue is diluted in a 100-µL volume to obtain a final percentage of Triton X-114 close to 10% (w/v). To test the performance of the overall method, linearity and precision were studied in deionized water samples under the optimum conditions described above. The experimental relationships between normalized peak area with internal standard (NPAis) and triazine concentration were found to be linear over the whole range studied (Table 2). The precision of the method, evaluated as relative standard deviation (RSD) over six consecutive days and four replicates per day, remained at acceptable values both in regard to migration times (RSD around 5%) and the values of NPAis (RSD between 7 and 12%). The effective concentration factor (NPA of peak after CPE step/NPA of peak without CPE) for methylthiotriazines is about 25-42. In the case of the chlorotriazines, owing to their lower hydrophobicity, this lies between 7, for simazine, and 19, for propazine. Determination of Triazines in Waters. The procedure was applied to determine triazines in spiked tap and river water samples. Nonspiked samples did not reveal the presence of any of the triazines studied. The data offered in Table 3 reflect

Table 2. Analytical Characteristics of the Nonaqueous CZE Method for the Determination of Triazines after CPE regression parameters NPAis vs concentrationa (mol L-1) slope Am Tb Pm Sz Az Pz

0.608 ( 0.007 1.65 ( 0.02 1.32 ( 0.03 0.32 ( 0.01 0.70 ( 0.01 1.43 ( 0.02

intercept ×

108

1.3 ( 1.0 -3.3 ( 2.6 4.1 ( 4.5 11.7 ( 7.9 5.1 ( 4.4 -5.9 ( 6.3

day to day RSD (%)b r2 0.997 0.997 0.986 0.973 0.994 0.995

range (µg

L-1)

25-500 26-510 26-520 109-2180 66-1310 51-1020

migration times (min)

NPAis

concn lev.c (µg L-1)

3.4 3.5 3.7 4.0 4.7 5.1

8.1 8.6 7.0 9.8 11.2 11.4

100 102 104 436 262 204

a Normalized peak area internal standard () NPA b c triazine/NPAinternal standard). Six consecutive days. Four replicates each day. Concentration level of each triazine for RSD calculations.

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Table 3. Determination of Triazines from River and Tap Water Samples by the Nonaqueous CZE Method after CPE found (µg L-1)a triazine

added (µg L-1)

river water

tap water

ametryne terbutryne prometryne simazine atrazine propazine

186 153 149 675 372 299

195 ( 15 145 ( 13 142 ( 10 716 ( 35 396 ( 31 280 ( 20

193 ( 14 148 ( 13 147 ( 11 651 ( 31 382 ( 26 289 ( 22

a

For n ) 3.

acceptable predictions for all the triazines. On the basis of these results it should be possible to determine triazines in this type of matrix. CONCLUSIONS The results obtained reveal that the use of nonaqueous media in the separation buffer permits the electrophoretic separation of samples with high-surfactant contents, avoiding the adsorption of the surfactant onto the walls of the capillary. This type of sample can be generated either after a preconcentration step that uses the cloud point methodology or may come from environmental sources that have been treated with surfactants25 to increase the rate of remediation of soils and waters contaminated by nonpolar organic pollutants. (25) Smith, J. A.; Sahoo, D.; Mclellan, H. M.; Imbrigiotta, T. E. Environ. Sci. Technol. 1997, 31, 3565-3572.

2474 Analytical Chemistry, Vol. 71, No. 13, July 1, 1999

Different authors have already reported the advantages of using nonaqueous media in capillary electrophoresis.26-29 In this sense, the use of nonaqueous media enables the cloud point extraction to be employed as a preconcentration step prior to CE analysiss yet another advantage. Moreover, modification of the concentration of perchloric acid in the medium in which the sample is injected may be used to determine the pKa* of chlorotriazines in nonaqueous media. The results obtained in the analysis of different spiked water samples indicate that CPE can be satisfactorily used as a preconcentration step prior to the analysis of triazines by capillary electrophoresis. ACKNOWLEDGMENT The Direccio´n General de Investigacio´n Cientı´fica y Te´cnica (DGICYT, Spain, Project PB95-1000) and the Consejerı´a de Educacio´n y Ciencia de la Junta de Castilla y Leo´n (Project SA 29-96) are gratefully acknowledged for financial support for this work. SUPPORTING INFORMATION AVAILABLE MECC separation of triazines in the presence of SDS and CTAB after cloud point extraction. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review November 9, 1998. Accepted March 11, 1999. AC9812227 (26) Walbroehl, Y.; Jorgenson, J. W. J. Chromatogr. 1984, 315, 135-143. (27) Walbroehl, Y.; Jorgenson, J. W. Anal. Chem. 1986, 58, 479-481. (28) Lu, W.; Poon, G. K.; Carmichael, P. L.; Cole, R. B. Anal. Chem. 1996, 68, 668-674. (29) Wang, F.; Khaledi, M. G. Anal. Chem. 1996, 68, 3460-3467.