Anal. Chem. 1997, 69, 4437-4444
Capillary Zone Electrophoresis in Nonaqueous Solvents in the Presence of Ionic Additives 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
Capillary zone electrophoresis (CZE) in nonaqueous media and in the presence of ionic additives has been successfully applied to the determination of compounds that differ only slightly in their electrophoretic mobilities. Triazine herbicides of environmental interest were chosen as test compounds because they behave as very weak bases. CZE separation of these analytes (especially chlorotriazines) in aqueous solution is difficult due to the low pH required for their conversion into protonated cationic form (HA+). However, in mixed nonaqueous solvents, 50% (v/v) acetonitrile-methanol, the acid-base characteristics of these compounds are modified, yielding the protonated ionic species that is susceptible to migration when subjected to an electric field. A noteworthy increase in separation selectivity and resolution can be achieved by using ionic additives. Thus, in this mode of capillary zone electrophoresis, separation is based on ionic interactions between the charged analytes and the ionic additive present in the separation medium. These interactions contribute to enhancing mobility differences and to improving analyte separation. For the separation of chloroand methylthiotriazines, 10 mM perchloric acid in 50% (v/v) acetonitrile-methanol and 20 mM SDS proved to be satisfactory, providing high resolution in short analysis times. The selectivity achieved was found to depend on the degree of association of the analyte with the ionic additive in the nonaqueous medium. This permits manipulation of the selectivity of the electrophoretic separations as a function of the type and concentration of the ionic additive and of the nature of the nonaqueous medium employed. The use of nonaqueous media in capillary electrophoresis (CE) is a recently developed field, although, in more classic modes of electrophoresis, application of organic media is a widespread practice.1-3 The use of nonaqueous media and of binary water-organic solvent systems extends the range of application of CE, rendering electrophoretic separations more versatile since it is possible to work in media with different dielectric constants, polarities, densities, viscosities, and acid-base properties. Initially, the use of nonaqueous media in CE focused on their use as organic modifiers4-7 for the analysis of sparingly water(1) Hayek, M. J. Phys. Colloid. Chem. 1951, 55, 1527-1533. (2) Tuckerman, M. M.; Strain, H. H. Anal. Chem. 1960, 32, 695-698. (3) Parekh, N. J.; Fatmi, A. A.; Tshabalala, M. A.; Rodgers, L. B. J. Chromatogr. 1984, 314, 65-82. (4) Fujiwara, S.; Honda, S. Anal. Chem. 1987, 59, 487-490. (5) Tomlinson A. J.; Benson L. M.; Landers, J. P.; Scanlan, G. F.; Fang, J.; Gorrod, J. W.; Naylor, S. J. Chromatogr. 1993, 652, 417-426. S0003-2700(96)01303-0 CCC: $14.00
© 1997 American Chemical Society
soluble compounds and of compounds that show very similar mobilities in aqueous media. The first work to describe a CE application in a pure nonaqueous solvent was that of Walbroehl and Jorgenson8 for the separation of quinoline-like compounds in acetonitrile. Since then, interest in research into the possibilities offered by CE in nonaqueous systems or in binary aqueous/organic solvent systems has increased considerably. Theoretical aspects such as the effect of organic solvents on the electroosmotic mobility and the ζ-potential have been studied,9,10 and the use of certain nonaqueous solvents such as formamide11 as candidate solvents for capillary electrophoresis has been evaluated. Analytical applications of nonaqueous CE have been described for the separation of small inorganic anions12 and ionic hydrophobic solutes such as anionic surfactants,13 phenols,14 or positively chargeable drugs.15 The use of nonaqueous solvents also permits electrophoretic separation of electrically neutral compounds. In this sense, CZE separations of nonionic organic compounds have also been described.16,17 This type of separation is based on the interaction of neutral solutes and ionic buffer additives such as tetraalkylammonium ions dissolved in aqueous acetonitrile, forming a positively charged species that can migrate in an electric field. Other aspects related to the use of organic media in CE are that it improves the sensitivity in CE-MS,18,19 and also permits modification of the separation conditions of chiral species.20 In the present work, we describe a new way of increasing the separation selectivity in CZE in nonaqueous medium based on ion-pairing interactions between an analyte and an ionic additive added to the separation medium. To accomplish this, some of the characteristics offered by organic solvents over aqueous media are exploited: (a) their different acid-basic chemistry and (b) the possibility of the formation of ionic associations between (6) Benson L. M.; Tomlinson A. J.; Reid, J. M.; Walker, D. L.; Ames, M. M.; Naylor, S. J. High Resolut.Chromatogr. 1993, 16, 324-326. (7) Tomlinson A. J.; Benson L. M.; Naylor, S. J. High Resolut. Chromatogr. 1994, 176, 175-177. (8) Walbroehl, Y.; Jorgenson, J. W. J. Chromatogr. 1984, 315, 135-143. (9) Schwer, C.; Kenndler, E. Anal. Chem. 1991, 63, 1801-1807. (10) Schu ¨ lzner, W.; Kenndler, E. Anal. Chem. 1992, 64, 1991-1995. (11) Sahota, R. S.; Khaledi, M. G. Anal. Chem. 1994, 66, 1141-1146. (12) Salimi-Moosavi, H.; Cassidy, R. M. Anal. Chem. 1995, 67, 1067-1073. (13) Salimi-Moosavi, H.; Cassidy, R. M. Anal. Chem. 1996, 68, 293-299. (14) Masselter, S. M.; Zemann, A. J. Anal. Chem. 1995, 67, 1047-1053. (15) Zhang, C.-X.; von Heeren, F.; Thormann, W. Anal. Chem. 1995, 67, 20702077. (16) Walbroehl, Y.; Jorgenson, J. W. Anal. Chem. 1986, 58, 479-481. (17) Shi, Y.; Fritz, J. S. J. High Resolut. Chromatogr. 1994, 17, 713-718. (18) Tomlinson, A. J.; Benson, L. M.; Naylor, S. LC-GC 1995, 8, 210-216. (19) Lu, W.; Poon, G. K.; Carmichael, P. L.; Cole, R. B. Anal.Chem. 1996, 68, 668-674. (20) Wang, F.; Khaledi, M. G. Anal. Chem. 1996, 68, 3460-3467.
Analytical Chemistry, Vol. 69, No. 21, November 1, 1997 4437
Table 1. Chemical Structures and pKa Values in Water for the Triazines Studied
name
R1
ametryne terbutryne prometryne
S-CH3 S-CH3 S-CH3
simazine atrazine propazine
Cl Cl Cl
a
R2
R3
Methylthiotriazines NH-CH(CH3)2 NH-CH2-CH3 NH-CH2-CH3 NH-C(CH3)3 NH-CH(CH3)2 NH-CH(CH3)2 Chlorotriazines NH-CH2-CH3 NH-CH(CH3)2 NH-CH(CH3)2
NH-CH2-CH3 NH-CH2-CH3 NH-CH(CH3)2
pKaa 4.00 4.38 4.05 1.65 1.68 1.85
Data from ref 34.
charged compounds to form ion pairs, which is favored in media with low dielectric constants. Some evidence of ion association effects that lead to changes in selectivity has been reported by Salimi-Moosavi and Cassidy12,13 in the application of nonaqueous CE to the separation of inorganic anions and long-chain surfactants. However, these authors failed to find any analytical utility because, although the formation of ion pairs does produce changes in migration times, this does not afford any appreciable improvement in the resolution of adjacent peaks. Zhang et al.15 reported that ionic buffer additives that can form ion pairs with solutes were unsuitable to improve the separation of hydrophobic, positively chargeable compounds. Triazine herbicides were chosen as test compounds due to their environmental relevance as pollutants. Generally, triazines are weak bases that become protonated at low pH values, depending on their substituents (Table 1), to give the cationic form (HA+). The chlorotriazines (atrazine, propazine, simazine) have a much weaker basic nature than methylthiotriazines (ametryne, prometryne, terbutryne). The pKa values of chlorotriazines are about 1.6, whereas those of methylthiotriazines are close to 4. Several procedures employing CZE have been proposed21,22 for the determination of methylthiotriazines. However, these methods are not suitable for the electrophoretic separation of chlorotriazines because such species require a strongly acidic medium for their conversion into the cationic form. Accordingly, chlorotriazines are mainly separated as neutral species by MECC.23-26 In this work, we report that chloro- and methylthiotriazines can be simultaneously separated by CZE in mixed nonaqueous solvents when a suitable ionic additive is added to the electrolyte. The migration behavior of a mixture of six triazines in several nonaqueous media was studied. The use of ionic buffer additives that can form ion pairs with the solutes is shown to be an appropriate way of improving the electrophoretic separation of these compounds. The results obtained also suggest that the use (21) Foret, F.; Sustacek, V.; Bocek, P. Electrophoresis 1990, 11, 95-97. (22) Cai, J.; El-Rassi, Z. J. Liq. Chromatogr. 1992, 15, 1179-1192. (23) Cai, J.; El-Rassi, Z. J. Chromatogr. 1992, 608, 31. (24) Desiderio, C.; Fanalli, S. Electrophoresis 1993, 13, 698. (25) Dinelli, G.; Bonetti, A.; Catizone, P.; Galletti, G. C. J. Chromatogr. B 1994, 656, 275. (26) 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. 1996, 733, 349-360.
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of organic media and ionic additives may be a useful approach to optimize the separation of other ions that differ only slightly in their electrophoretic mobilities. EXPERIMENTAL SECTION Apparatus. Capillary electrophoresis was performed with a P/ACE 2000 (Beckman Instruments, Fullerton, CA) equipped with a UV detector. Standard P/ACE capillaries were used: 75 µm i.d., 57 cm long, detection at 50 cm. Reagents. All triazine herbicides were obtained from Riedel de Hae¨n (Seelze-Hannover, Germany) and were used without further purification (minimum purity greater than 98%). The chlorotriazines studied were as follow: atrazine, 2-chloro-4(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 as follow: 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. The organic solvents, acetonitrile (AcN) and methanol (MeOH), were of HPLC grade and used as received. Sodium dodecyl sulfate (SDS) was obtained from Fluka (Buchs, Switzerland); sodium dioctyl sulfosuccinate (DOSS) was from Aldrich (Madrid, Spain); tetrabutylammonium chloride (TBACl) was from Sigma (Madrid, Spain); and lithium chloride, lithium perchlorate, and perchloric acid were from Panreac (Barcelona, Spain). Tetrabutylammonium perchlorate (TBAClO4) was prepared with tetrabutylammonium chloride and perchloric acid: a known amount of TBACl is dissolved in water with a small percentage of methanol to ensure complete dissolution. Perchloric acid is added to this solution until a precipitate appears, followed by a small excess. The precipitate thus formed is filtered and washed with a watermethanol mixture until the washing liquid gives a negative acidic reaction. All other chemicals used for the preparation of the buffer electrolytes were of analytical reagent grade. Procedure. Uncoated capillaries were used throughout the study. All new capillaries were conditioned before use. They were pretreated sequentially for 5 min with water, 5 min with 0.5 M sodium hydroxide, a further 5 min with water, 5 min with methanol, and 5 min with the separation buffer. The daily startup procedure was 5 min with methanol and 5 min with the separation buffer. No changes in migration times were observed on a runto-run basis. 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. Unless otherwise stated, analysis was performed with an applied voltage of 22 kV, with the capillary thermostated at 25 ( 0.1 °C and with UV detection at 214 nm. The running buffer was 10 mM perchloric acid in different solvents (water, acetonitrile, methanol, or binary mixtures of these) and in the presence of different ionic additives at varying concentrations. The optimum separation medium was 50% (v/v) acetonitrile-methanol, 10 mM perchloric acid, and 20 mM SDS. Stock solutions of each triazine were prepared in acetonitrile at 500 µg/mL. A standard mixture of all six triazines at 5 µg/mL each and 2 mM HClO4 was prepared in acetonitrile. Electrolyte solutions of perchloric acid in pure acetonitrile and 50% (v/v) acetonitrile-methanol were prepared fresh.
Calculation of Electrophoretic Mobility. The electrophoretic mobility of the solutes was calculated according to the expression
µep ) µobs - µeo )
[
]
LtLd 1 1 V tM teo
(1)
where µobs is the observed overall mobility, µeo is the electroosmotic mobility, tM is the migration time of the solute, teo is the migration time for an uncharged solute (electroosmotic flow marker), Ld is the capillary length between the injection and the detection window, Lt is the total capillary length, and V is the applied voltage. The disturbance in the baseline due to the acetonitrile was used as the marker for the electroosmotic flow. In the experiments carried out at pH* ) 2, the value of electroosmotic mobility, µeo, was considered to be negligible (µep ≈ µobs). Measurement of pH* and Calculation of pKa* in 50% (v/ v) Acetonitrile-Methanol Medium. Since in aqueous and nonaqueous solutions pH may not be compared directly, henceforth the term apparent pH, or pH*, will be used. The values of pH* were determined by potentiometric measurements carried out using a glass electrode previously calibrated with solutions of known [H+] . To do so, solutions of perchloric acid at concentrations ranging between 10-3 and 10-2 M were employed because this acid behaves as a strong acid in acetonitrile and methanol.27,28 The concentration of the stock solution of perchloric acid was determined by standardization with potassium hydrogen phthalate.28,29 The determination of the pKa* for an acid species (HA+) was accomplished using the experimental data on the observed overall mobility (µobs) and on the electroosmotic flow (µeo) as a function of pH*. The migration behavior of a weak acid HA+ can be described by the following relationship:
[H+]* µobs - µeo ) µHA+ + [H ]* + Ka*
(2)
where (µobs - µeo) is the electrophoretic mobility at a given pH*, µHA+ is the electrophoretic mobility of the protonated (cationic) form, and Ka* is the dissociation constant of the acid HA+. This expression is similar to that deduced by Smith and Khaledi30 to describe the migration behavior of a weak acid (HA) in CZE. As a function of experimental parameters such as tM and teo, eq 2 can be written as
[
]
1 1 tM teo
-1
)
LtLd LtLd 1 + Ka* VµHA+ VµHA+ [H+]*
(3)
The slope of the plot of [(1/tM) - (1/teo)]-1 against 1/[H+]* permits the determination of the value of Ka* since the value of µHA+ is calculated from the intercept of the line. The experimental measurements of tM and teo as a function of pH* were made in (27) Kolthoff, I. M.; Bruckenstein, S.; Chantooni, M. K. J. Am. Chem. Soc. 1961, 83, 3927-3935. (28) Coetzee, J. F.; Kolthoff, I. M. J. Am. Chem. Soc. 1957, 79, 6110-6115. (29) Fritz, J. S. Anal. Chem. 1953, 25, 407-411. (30) Smith, S. C.; Khaledi, M. G. Anal. Chem. 1993, 65, 193-198.
50% (v/v) acetonitrile-methanol medium in the presence of both systems: acetic acid-sodium acetate (total concentration 100 mM) and perchloric acid-lithium perchlorate (total concentration 10 mM). RESULTS AND DISCUSSION CZE Separation of Triazines in Aqueous Medium. The problems associated with CZE separation of chloro- and methylthiotriazines in aqueous systems are illustrated in Figure 1a. The figure shows the electropherogram obtained in 10 mM aqueous perchloric acid when a mixture of six triazines (three chloro- and three methylthiotriazines) was injected. Only three peaks can be observed in Figure 1a: the first corresponds to the sum of ametryne and terbutryne; the second to prometryne; and the third, appearing at times greater than 30 min, corresponds to the three chlorotriazines injected: atrazine, simazine, and propazine. These results show that, in 10 mM HClO4 aqueous medium, it is not possible to perform CZE separation of chlorotriazines; due to their low pKa value (Table 1), only a small fraction is found in protonated form (HA+), and hence all the chlorotriazines migrate in a group close to the electroosmotic flow, which at these pH values was very low. In this aqueous medium, separation of the methylthiotriazines was also deficient. In 10 mM HClO4, all the methylthiotriazines would be expected to be found in acid cationic form (HA+) (Table 1); however, the differences in their electrophoretic mobilities in aqueous medium are insufficient to obtain suitable resolution among them. When acid electrolytes of higher concentration are usedsabout 100 mMshigh current intensities are obtained, together with strong fluctuations and frequent drops in current. This invalidates the analysis. These findings are consistent with earlier reports26 in which other acid media were assayed. Effect of the Nonaqueous Solvent in CZE Separation of Triazines. When 10 mM HClO4 in 100% acetonitrile was used as the separation medium, the electropherogram shown in Figure 1b was obtained: still, only five separated peaks were observed from an injection of the mixture of six triazine standards. However, the migration time at which the three chlorotriazines appearedsabout 5 minsis considerably lower than that observed in aqueous medium (30 min). By contrast, the migration times of the peaks corresponding to the methylthiotriazines (about 4.5 min) undergo a smaller decrease than those of the chlorotriazines (Figure 1a,b). A more detailed study of the migration behavior of chloro- and methylthiotriazines for electrolyte solutions containing 0-100% acetonitrile is illustrated in Figure 2, which shows the variation in electrophoretic mobility as a function of the percentage of acetonitrile in the separation medium. Propazine and ametryne were selected as representative herbicides of both groups, chloroand methylthiotriazines, respectively. The experimental values of electrophoretic mobility (Figure 2, curves 1 and 2, for ametryne and propazine, respectively) were compared with the values calculated taking into account only the variation in the viscosity of the separation medium on modifying the content of acetonitrile. The expression used was
µep(mixture) ) µep(water)[ηwater/ηmixture] Analytical Chemistry, Vol. 69, No. 21, November 1, 1997
(4) 4439
Figure 1. Electropherograms for a mixture of chloro- and methylthiotriazines. Separation electrolyte, 10 mM perchloric acid in (a) water, (b) 100% acetonitrile, (c) 100% methanol, and (d) 50% (v/v) acetonitrile-methanol. Sample concentration, 5 µg/mL of each triazine in 100% acetonitrile and 2 mM perchloric acid. (a) Hydrodynamic injection, 5 s; (b)-(d) electrokinetic injection at 5 kV for 15 s. Applied voltage, 22 kV. Peaks: (1) ametryne, (2) terbutryne, (3) prometryne, (4) simazine, (5) atrazine, and (6) propazine.
Figure 2. Dependence of electrophoretic mobility on the content of acetonitrile in water. Experimental data (curves 1 and 2) and calculated values (curves 3 and 4) for ametryne (Am) and propazine (Pz). Hydrodynamic injection, 5 s. Separation electrolyte, 10 mM perchloric acid. Other conditions are as described for Figure 1.
where µep(water) is the experimental value determined in 100% aqueous medium, ηwater is the viscosity of the water, and ηmixture are values given in the literature for water-acetonitrile mixtures.31 The electrophoretic mobilities calculated according to eq 4 are shown in curves 3 and 4 of Figure 2. For acetonitrile percentages 4440
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lower than 50%, a consistent trend is seen between the experimental and calculated values, although, considering only the effect of viscosity, the former are generally lower than those calculated according to eq 4. This behavior may be related to the variation in the dielectric constant of the medium. In water-acetonitrile mixtures, the dielectric constant decreases in an approximately linear fashion with the increase in the acetonitrile content.9,31 This must imply a decrease in the mobility of the ionic solutes due to the effect of this parameter on the effective ionic radius and on the fraction of ion pairs formed. In water-acetonitrile mixtures with acetonitrile contents above 30% (v/v) and below 90% (v/v), it was not possible to obtain precise experimental measurements of the mobilities of the chlorotriazines; this was because, in these media, the mobilities of these analytes are so low that they do not appear in analysis times shorter than 80 min. For high percentages of acetonitrilesabove 90%sa sharp increase in the mobility of the chlorotriazines is observed (curve 2 of Figure 2). This increase in the mobility of the chlorotriazines in media with high acetonitrile contents seems to be related to a modification of the acid-base properties of these compounds. In pure acetonitrile, chlorotriazines migrate at a velocity comparable to that of the methylthiotriazines, suggesting that, in this medium, all the triazines are in protonated HA+ form. (31) Janz, G. J.; Tomkins, R. P. T. Nonaqueous Electrolytes Handbook; Academic Press, Inc.: New York, 1973; Vol. I.
Table 2. Electroosmotic Mobility in 10 mM Perchloric Acid for Different Separation Media separation medium
µeo (V-1 cm2 s-1) × 105
H2O
AcN
MeOH
AcN/MeOH 50% (v/v)
6.8
7.2
4.3
6.5
where µep(acetonitrile) is the experimental value determined in 100% acetonitrile, ηacetonitrile is the viscosity of acetonitrile, and ηmixture represents the data reported in the literature for the viscosity of acetonitrile-methanol mixtures.31 The electrophoretic mobilities calculated according to eq 5 are shown in curves 3 and 4 of Figure 3.
In the case of ametryne, the experimental results show reasonable agreement with those calculated as a function of the variation in the viscosity of the medium (curves 1 and 3 in Figure 3). In acetonitrile-methanol mixtures, the dielectric constant of the medium also undergoes an approximately linear decrease with the increase in methanol.31 In this case, the variation is less marked due to the magnitude of the values of the dielectric constant in acetonitrile ( ) 36.01) and in methanol ( ) 32.63). For propazine (a chlorotriazine), the experimental mobility values are lower than the calculated ones, this difference being more pronounced when the proportion of methanol was higher. Thus, in 100% methanol, the mobility of propazine is considerably lower than that expected in pure methanol. This marked decrease in the electrophoretic mobility of the chlorotriazines points to a decrease in the effective charge on the analyte, caused by an increase in the basicity of the medium when changing from acetonitrile to a solvent of greater basicity, such as methanol. Figure 1c shows the electropherogram obtained in 100% methanol; it may be seen that the increase in the migration time of the chlorotriazines is some 5-7 min with respect to that obtained in 100% acetonitrile medium (Figure 1b). The methylthiotriazines undergo shorter delayssabout 2 min. In this 100% methanol medium, the intensity of the electric current is highly unstable and cannot be reproduced. For acetonitrile-methanol mixtures of intermediate composition, an appreciable improvement in the stability of the current is seen, together with good resolution in the separation of the six triazines due to the addition of the second solvent. Figure 1d shows the electropherogram obtained when 10 mM HClO4 in 50% (v/v) acetonitrile-methanol was used. A more efficient separation of the chlorotriazines is observed with respect to that obtained in pure acetonitrile. The behavior of the electroosmotic flow was also examined for electrolyte solutions containing 10 mM HClO4 in aqueous medium and in medium containing 100% acetonitrile, 100% methanol and 50% (v/v) acetonitrile-methanol (Table 2). In 10 mM HClO4 in 50% (v/v) acetonitrile-methanol, the electroosmotic mobility was slightly lower than that obtained in pure aqueous medium. This shows that, in these media of low pH,33 electroosmotic flow becomes insignificant, and hence its contribution to the overall mobility is negligible. Accordingly, the mobility observed in this medium can be said to be almost entirely due to electrophoretic mobility. Effect of pH*. In the electrophoretic separation of ionizable compounds, the value of the pH of the separation medium plays an important role since it determines the extent of ionization of each individual compound. In nonaqueous media, it is more appropriate to use the term pH* to indicate the activity of hydrogen ions in the medium. In this study, the pH* value was determined from potentiometric measurements, as described in the Experimental Section.
(32) Kilpatrick, M., Jr.; Kilpatrick, M. L. Chem. Rev. 1933, 33, 131-134.
(33) Lambert, W. J.; Middelton, D. L. Anal. Chem. 1990, 62, 1585-1587.
Figure 3. Dependence of electrophoretic mobility on the content of methanol in acetonitrile. Experimental data (curves 1 and 2) and calculated values (curves 3 and 4) for ametryne (Am) and propazine (Pz). Electrokinetic injection at 5 kV for 15 s. Separation electrolyte, 10 mM perchloric acid. Other conditions are as described for Figure 1.
Perchloric acid is completely dissociated27,28 in acetonitrile. In this solvent, reaction between the solvated proton and even extremely weak bases is more complete than that in water. Kilpatrick and Kilpatrick32 established general relationships between the pK values in acetonitrile (pKCH3CN) and in aqueous medium (pKH2O) as a function of the type of acid. For cationic acids of the amine type, pKCH3CN ) 0.47pKH2O + 2.2. According to this expression, the values of pKCH3CN for the chlorotriazines would be approximately 3. Thus, in a medium with pH* ) 2, (10 mM HClO4 in acetonitrile), these analytes would mainly be in protonated form. This would explain the sharp increase in their migration mobilities in media with percentages of acetonitrile greater than 90%. CZE Separation of Triazines in Acetonitrile-Methanol Mixtures. Since, in 100% acetonitrile, the experimentally determined electrophoretic mobilities of both types of triazine are very similar, the electropherogram obtained in this medium has low resolution (Figure 1b). Addition of a second organic solvent may alter the electrophoretic behavior of the analytes and affect the selectivity of separation because it modifies the behavior of the ionic species in solution. Methanol was employed as a second solvent. The electrophoretic mobilities of ametryne and propazine were determined as a function of the methanol content of the electrolyte solution, 10 mM HClO4 in acetonitrile (curves 1 and 2 in Figure 3). As well as the experimental measurements, the mobility values were calculated considering only the variation in viscosity of the acetonitrile-methanol mixtures:
µep(mixture) ) µep(acetonitrile)[ηacetonitrile/ηmixture]
(5)
Analytical Chemistry, Vol. 69, No. 21, November 1, 1997
4441
Figure 4. Electrophoretic and electroosmotic mobilities as a function of pH* in 50% (v/v) acetonitrile-methanol. Hydrodynamic injection, 5 s. Other conditions are given in the Experimental Section.
In order to check the effect of the separation medium on the acid-base properties of the triazines, the electrophoretic mobilities of ametryne and propazine were evaluated experimentally as a function of pH*. As may be seen in Figure 4, mobility increased when the pH* decreased because of the increase in the effective charge on the analyte. At sufficiently acidic pH* values, mobility tends toward a constant maximum value, indicating that the analytes are completely protonated. The point of inflection of this curve can be interpreted as the pKa* value of the acid cationic form HA+. Estimation of the pKa* values for ametryne and propazine according to the procedure described in the Experimental Section (eq 3) yielded pKa* values of 6.1 for ametryne and 3.2 for propazine. These values are considerably higher than those reported in the literature for aqueous media34 and show that, under the experimental conditions employedsin 10 mM HClO4, 50% (v/ v) acetonitrile-methanolsboth types of triazine are in their protonated form. Effect of Ionic Additives. Use of this 50% (v/v) acetonitrilemethanol organic medium permitted a substantial improvement in the electrophoretic separation of chloro- and methylthiotriazines with respect to the separation obtained in aqueous medium (Figure 1a and d). However, the resolution of the separation was not complete. This may be due to the similarity in the chemical structure of these analytes, which does not allow electrophoretic mobilities sufficiently different to obtain a fully resolved electropherogram. One relevant characteristic of nonaqueous solvents with low dielectric constants ( < 40) is the importance acquired by the formation of ion pairs in the bulk solution since the intensity of the electrostatic interactions between ions of opposite charges increases inversely to the value of the dielectric constant. The neutral aggregates formed no longer participate in the conductivity nor in the ionic strength of the solution and behave as molecular solutes. The possibility of using interactions between a charged analyte and an ionic species added to the separation medium to introduce additional differences in electrophoretic mobilities was assessed. To do so, we studied the effect of six ionic additives on the electrophoretic mobilities of chloro- and methylthiotriazines: sodium dodecyl sulfate (SDS), sodium dioctyl sulfosuccinate (34) Weber, J. B. Residue Rev. 1970, 32, 93-130.
4442 Analytical Chemistry, Vol. 69, No. 21, November 1, 1997
Figure 5. Effect of the type and concentration of additive on the electrophoretic mobility of (a) ametryne and (b) propazine. Sample concentration, 5 µg/mL of each triazine in 100% acetonitrile and 2 mM perchloric acid. Separation electrolyte, 10 mM perchloric acid in 50% (v/v) acetonitrile-methanol. Electrokinetic injection at 15 kV for 5 s.
(DOSS), tetrabutylammonium perchlorate (TBAClO4), tetrabutylammonium chloride (TBACl), lithium perchlorate (LiClO4), and lithium chloride (LiCl). Figure 5, parts a and b, shows the variation in electrophoretic mobility of ametryne and propazine, respectively, as a function of the concentration of each of the additives studied. Although all the additives produced a decrease in the migration mobility of both types of triazine, the magnitude of this effect differed according to the type of additive used. Thus, whereas LiCl led to only slight decreases in mobility, the greatest effects were found for SDS and DOSS, for which the association was produced to a greater extent. The degree of association of the perchlorate anions was lower than that of the anionic surfactants and similar to that of the chlorides. The effect of the additives on the decrease in electrophoretic mobility decreases in the following order: SDS, DOSS > ClO4-, Cl-. Figure 6 shows the experimental measurements of current intensity generated in 50% (v/v) acetonitrilemethanol medium, 10 mM HClO4 in the presence of different concentrations of the ionic additives. It should be noted that the electrolyte used, 10 mM HClO4, already introduces a concentration of perchlorate anions into the medium, such that, even in the absence of other additives, it may be assumed that a certain degree of ionic association is present. In view of the results obtained, it is possible to propose the existence of a dynamic equilibrium involving the participation of
Table 3. Calculated tep and K Values for the Triazines Studied Ka triazine ametryne terbutryne prometryne simazine atrazine propazine a
Figure 6. Effect of the type and concentration of additive on the current intensity generated. Experimental conditions are as described for Figure 5.
the cationic protonated form of the triazines HA+ and the anions present in solution: the ClO4- anion and the S- anion coming from the additives. These equilibria can be represented thus:
tep/min TBAClO4 LiClO4 TBACl LiCl SDS DOSS 4.13 4.13 4.33 4.54 4.68 4.74
0.41 0.41 0.38 0.41 0.41 0.43
0.48 0.48 0.45 0.51 0.51 0.55
0.53 0.61 0.54 0.59 0.65 0.75
0.30 0.36 0.29 0.32 0.36 0.43
0.81 0.85 0.87 0.82 0.90 1.01
0.93 1.00 1.01 0.97 1.10 1.27
Values for 30 mM of additive + 10 mM of perchloric acid.
Expression 11 is similar to that obtained by Khaledi et al.35 to describe the migration behavior of cationic compounds in MECC, but with the particularity that, in our case, there is no micellar phase and that, at the working pH*, one has [HA+] . [A]. In the presence of only the perchlorate anion, expression 11 becomes
1 µep ) µHA+ 1 + K1
(12)
In 50% (v/v) acetonitrile-methanol medium, 10 mM HClO4 (pH* ) 2), and in the presence of additives at a concentration of 30 mM, the electroosmotic flow is considered to be negligible since its signal should appear at times greater than 60 min. Under these conditions, it is assumed that µep ) µobs - µeo ≈ µobs. As a function of the experimental migration time, tM, expression 12 can be rewritten as
where
KIP1 )
[HA+ClO4-] [HA ][ClO4 ]
KIP2 )
+
(6)
tM ) tep + tepKIP1[ClO4-]
(7)
and the plot of tM against the perchlorate concentration allows the determination of the value of tep, the migration time in the absence of any anion able to form ion pairs with the analyte (Table 3). In the same way, knowing the value of tep, from expression 11 it is possible to obtain the value of the sum of K1 + K2:
-
[HA+S-] [HA+][S-]
The ratio between the number of associated and unassociated cations can be defined by K1 ) nHA+ClO4-/nHA+ and K2 ) nHA+S-/ nHA+. One thus has
K1 ) KIP1[ClO4-]
(8)
K2 ) KIP2[S-]
(9)
(10)
From expressions 8, 9, and 10, it may be deduced that
1 µep ) µHA+ 1 + K1 + K2
(14)
where K represents the relationship
Working at values of pH* < pKa*, the predominant form in solution is the protonated one, such that the equilibrium between this and the unprotonated form is not considered. In this working medium, one has that
nHA+ µep ) µobs - µeo ) µHA+ nHA+ + nHA+ClO4-+ nHA+S-
tM - tep ) K1 + K2 ) K tep
(13)
K)
nHA+ClO4- + nHA+SnHA+
)
nassociated nunassociated
(15)
Table 3 shows the estimated values of K for an additive concentration of 30 mM. These values indicate that the interactions among the protonated triazines and the anions studied are relatively weak and that they become stronger when anionic surfactants are employed. Figure 7 shows the effect of the concentration of SDS on the mobility of the six triazines assayed. It may be seen that a concentration 20 mM of SDS, at least, is necessary to obtain good
(11) (35) Strasters, J. K.; Khaledi, M. G. Anal. Chem. 1991, 63, 2503-2508.
Analytical Chemistry, Vol. 69, No. 21, November 1, 1997
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behavior of cationic and hydrophobic compounds in the presence of low amounts of SDS; with 1 mM SDS, all cationic drugs were found to form neutral ion pairs and coeluted with the solvent.
Figure 7. Dependence of electrophoretic mobility on the SDS concentration in the separation buffer. Other experimental conditions are as described for Figure 5.
Figure 8. Typical electropherogram for a mixture of chloro- and methylthiotriazines in 50% (v/v) acetonitrile-methanol and in the presence of 20 mM SDS. Experimental conditions are as described for Figure 5.
resolution (Figure 8). Use of higher SDS concentrations continues to produce a decrease in mobility, although with a less marked effect on resolution. The limited extent to which ionic association occurs has favorable effects on separation since extensive ion association would be expected to reduce the potential differentiation between analytes. Zhang et al.15 have examined the electrophoretic
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Analytical Chemistry, Vol. 69, No. 21, November 1, 1997
CONCLUSIONS In the present work, we describe a form of capillary zone electrophoresis in nonaqueous medium based on ionic interactions between the analyte and an ionic additive added to the separation medium. Use of nonaqueous media allows CZE separations that are not possible in aqueous solution. The nonaqueous solvents have acid-base properties that differ from those of water, in turn affecting the acid-base characteristics of the analytes themselves. Thus, nonaqueous solvents permit a wider range of compounds to be ionized through the use of acid-base chemistry. The presence of ionic additivessdodecyl sulfate, dioctyl sulfosuccinate, perchlorate, and chloridespermits more selective electrophoretic separations through ionic interaction between the analyte and the additive. The extent to which ionic association occurs between the cationic analyte and the anionic additive depends on the type and concentration of the additive in the nonaqueous medium. Accordingly, an additional parameter in the manipulation of the selectivity of electrophoretic separations is the modification of the nature and percentage of the organic solvents employed and also those of the ionic additives used. The current generated, and hence the Joule heating, is lower in 50% (v/v) acetonitrile-methanol medium than in aqueous solution for the same electrolyte, 10 mM HClO4. This makes it possible to use very high electric fields and media of higher ionic strength. As a result, greater efficiencies and lower analysis times can be achieved. Finally, the use of nonaqueous media permits electrophoretic separations to be carried out in media with high contents in nonionic surfactants; the applications of this type of electrophoretic separation are currently being studied. 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 Cultura de la Junta de Castilla y Leo´n (Project SA2996) are gratefully acknowledged for financial support of this work. Received for review December 31, 1996. Accepted August 13, 1997.X AC9613038 X
Abstract published in Advance ACS Abstracts, September 15, 1997.
