Anal. Chem. 2000, 72, 1441-1447
A Fluorescence Detection Scheme for Capillary Electrophoresis of N-Methylcarbamates with On-Column Thermal Decomposition and Derivatization Yuan Sheng Wu,† Hian Kee Lee,† and Sam F. Y. Li*,†,‡
Department of Chemistry and Institute of Materials Research and Engineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Republic of Singapore
This paper describes a fluorescence detection method for N-methylcarbamate (NMC) pesticides in micellar electrokinetic chromatography (MEKC) separation. Fulfillment of the fluorescence detection hinged on the discovery that quaternary ammonium surfactants (particularly cetyltrimethylammonium bromide, CTAB), besides serving as hydrophobic pseudophases in MEKC, are also capable of catalyzing the thermal decomposition of NMCs to liberate methylamine. Thus, a multifunctional MEKC medium consisting of borate buffer, CTAB, and derivatizing components (o-phthaldialdehyde/2-mercaptoethanol) was formulated, which allowed first normal MEKC separation, subsequent thermal decomposition, and finally in situ derivatization of NMCs. With careful optimization of the operation conditions, fluorescence detection of 10 NMC compounds was achieved, with column efficiencies typically higher than 50 000 and detection limits better than 0.5 ppm. The present work represents an unprecedented effort in capillary electrophoresis (CE), in which an intact capillary was consecutively utilized as chambers for separation, decomposition, derivatization, and detection, without involving any interfacing features. The success in the implementation of such a detection system resulted in strikingly simple instrumentation as compared with the traditional postcolumn fluorescence determination of NMCs by reversed-phase HPLC. Similar protocols should be workable in the determination of a wide range of pesticides and pharmaceuticals in CE formats. N-Methylcarbamates (NMCs) represent an important category of pesticides ever since its first introduction into the agrochemical market in the1950s. Formed by reacting methyl isocyanate with various oxime or phenolic derivatives, these compounds exhibit a broad-spectrum efficacy. Hence, they are used worldwide as insecticides, acaricides, nematocides, and molluscicides on a large number of crops. Due to their relatively biodegradable and less soil persistent nature, there have been few attempts to investigate their environmental fates in their early years of application. With more stringent regulations for pesticides being imposed and more sensitive detection methods available, there are growing interests in the study of environmental implications of NMCs. Among the various methods for monitoring NMCs in foodstuffs and environment bodies, chromatographic techniques have been † ‡
Department of Chemistry. Institute of Materials Research and Engineering.
10.1021/ac990928d CCC: $19.00 Published on Web 02/23/2000
© 2000 American Chemical Society
employed extensively.1-3 Gas chromatography (GC) was employed mainly during the 1960s and 1970s. A major challenge in the analysis of NMCs by GC is their thermal lability. Therefore, various precautionary measures had to be taken for curbing the thermal degradation of NMCs during GC analysis. These included performing derivatization,4-8 lowering column temperature, using short columns, and adopting programmed temperature vaporization.9,10 Since the 1970s, high-performance liquid chromatography (HPLC) has become the preferred choice for the determination of NMCs, because in this case, the thermal lability problem is obviated.11 Although normal-phase separations were successfully implemented in many cases,12,13 reversed-phase HPLC (RP-HPLC) has been much more popular,14-17 due mainly to its operational simplicity and better performance. An additional advantage of RPHPLC is its compatibility with aqueous samples, which allows oncolumn enrichment of NMCs in environmental analysis. In the early period of analysis of NMCs via RP-HPLC, the prevailing measurement method was UV detection, which gave detection limits ranging between 1 and 10 ppb in the presence of various preconcentration means.1,18 Such detectability is deemed inadequate in case of ultratrace analysis, especially with respect to the stringent European Community Directive for Drinking Water, in which pesticides at the level of 0.1 ppb need to be quantified. A breakthrough in the determination of NMCs occurred in 1977, when Moyer et al. introduced postcolumn fluorescence detection (1) McGarvey, B. D. J. Chromatogr. 1993, 642, 89-105. (2) Liska, I.; Slobodnik, J. J. Chromatogr., A 1996, 733, 235-258. (3) Yang, S. S.; Goldsmith, A. I.; Smetena I. J. Chromatogr., A 1996, 754, 3-16. (4) Fishbein, L.; Zielinski, W. J. Chromatogr. 1965, 20, 9-14. (5) Wien, R. G.; Tanaka, F. S. J. Chromatogr. 1977, 130, 55-63. (6) Holden, E. R. J. Assoc. Off. Anal. Chem. 1973, 56, 713-717. (7) Lawrence, J. F.; Lewis, D. A.; Mcleod, H. A. J. Chromatogr. 1977, 138, 143150. (8) Coburn, J. A.; Ripley, B. D.; Chan, A. S. Y. J. Assoc. Off. Anal. Chem. 1976, 59, 188-196. (9) Hall, R. C.; Harris D. E. J. Chromatogr. 1979, 169, 245-259. (10) Muszhat, L.; Aharonson, N. J. Chromatogr. Sci. 1983, 21, 411-414. (11) Frei, R. W.; Lawrence, J. F. J. Chromatogr. 1973, 83, 321-330. (12) Frei, R. W.; Lawrence J. F.; Hope J.; Cassidy, R. M. J. Chromatogr. Sci. 1974, 12, 40-45. (13) Lawrence, J. F. J. Agric. Food Chem. 1977, 25, 211-212. (14) Aten, C. F.; Bourke, J. B. J. Agric. Food Chem. 1977, 25, 1428-1430. (15) Krause, R. T. J. Chromatogr. 1979, 185, 615-624. (16) Miles, C. J.; Moye, H. A. Anal. Chem. 1988, 60, 220-226. (17) Bellar, T. A.; Budde, W. L. Anal. Chem. 1988, 60, 2076-2083. (18) Dekok, A.; Hiemstra, M. J. Chromatogr. 1992, 623, 265-276.
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of NMCs.19 In this method, the eluate from RP-HPLC was allowed to mix with sodium hydroxide solution and was then transferred into a heated reactor, where NMCs were hydrolyzed to generate methylamine, and subsequently, a stream of derivatizing agent (o-phthalaldehyde/2-mercaptoethanol) was pumped into the main flow, thus producing an adduct with strong fluorescence (hydroxyethylthio-2-methylisoindole). The fluorescence detection of NMCs offered at least 1 order of magnitude improvement in detection sensitivity with respect to UV detection.1 Currently, RPHPLC with postcolumn fluorescence detection is accepted as a standard protocol for the determination of NMCs by many official organizations, including the U.S. Environmental Protection Agency (EPA) and Association of Official Analytical Chemist (AOAC), due to its outstanding sensitivity and specificity. It should be pointed out that postcolumn fluorescence detection of NMCs is achieved through the use of rather complicated equipment; i.e., two additional pumping systems and two reaction chambers are required on top of a normal HPLC instrument. Moreover, as the system is operated at elevated temperature, back-pressure regulation is often a must to prevent boiling of the mobile phase. Some efforts had been devoted to simplify the above instrumental setup. Two good examples were the studies of de Kok and Hiemstra,18 and Nondek et al.20 in which they demonstrated that a heated catalytic bed made of strong anion-exchange resin (Aminex A-27) or magnesium oxide could be employed to replace sodium hydroxide to effect the decomposition of NMCs, thus eliminating one set of pumping devices. In our earlier studies, micellar electrokinetic chromatography (MEKC) was found to be a promising alternative for the determination of NMCs.21,22 Compared with RP-HPLC, better separation could be readily obtained, due mainly to the inherently high column efficiency of MEKC. Moreover, a variety of means had been shown to be effective in enhancing the separation performance, including changing the buffer ionic strength, manipulating the surfactant concentration, and adding various buffer modifiers (cyclodextrins, urea, organic solvent, etc.). In these investigations, on-column UV detection was employed. A well-known problem associated with this detection scheme is that the detection sensitivity in terms of concentration is compromised, since the usable optical path is limited by the inner diameter of the separation capillary. As a result, we experienced a great difficulty in realizing detection limits that matched the prevailing requirement of environment analysis. In fact, to enable the detection of the 0.1 ppb level of NMCs in drinking water, a multistep procedure involving solid-phase extraction, solvent evaporation, and oncolumn stacking, which contributed to sample enrichment of several thousandfold, was required.22 Obviously, to facilitate the measurement of low levels of NMCs from various demanding samples using the MEKC technique, a detection scheme with higher sensitivity and selectivity must be sought. Similar to the situation in RP-HPLC, pursuing fluorescence detection of NMCs is an attractive choice. Besides its intrinsic sensitivity and specificity, fluorescence detection on a capillary offers the possibility of boosting sensitivity through the use of (19) Moye, H. A.; Scherer, S. (20) Nondek, L.; Frei, R. W.; 141-150. (21) Wu, Y. S.; Lee, H. K.; Li, (22) Wu, Y. S.; Lee, H. K.; Li,
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J.; John, P. A. Anal. Lett. 1977, 1049-1054. Brinkman, U. A. Th. J. Chromatogr. 1983, 282, S. F. Y. J. Microcolumn Sep. 1998, 10, 239-247. S. F. Y. J. Microcolumn Sep. 1998, 10, 529-535.
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laser-induced fluorescence detection (LIF). Unfortunately, unlike in HPLC, it is not a simple task to perform postcolumn derivatization in CE format,23,24 since a minute stream of derivatizing agent must be effectively introduced into the separation capillary with an inner diameter typically less than 75 µm. So far, in the realm of CE study, several interfacing structures have been created to enable the mixing of derivatizing solution with the separation flow. On the basis of their different configurations, these interfacing structures can be classified as coaxial reactor,25 sheath flow reactor,26 gap reactor27 and free solution reactor.28 Though with different degrees of success in practice, these interfacing structures not only were major sources of band broadening but also necessitated complicated microfabricaiton and micromanipulation. Furthermore, it was often hard to maintain the detection characteristic once the microreactor was changed, because it was not easy to ensure exactly the same alignment status for two microreactors. In the case of analysis of NMCs by MEKC, to realize postcolumn derivatization/fluorescence detection of NMCs, the analytes must be first decomposed with alkaline under elevated temperature and then derivatized with o-phthaldiadehyde (OPA) and 2-mercaptoethanol. It follows that the implementation of postcolumn fluorescence detection here would require the use of two microreactors to cope with the two separate chemical processes. Apparently, tremendous technical difficulties would be involved. On-column derivatization and fluorescence detection is a relatively new development in the arena of CE.29 Here the sample and its derivatizing reagent are injected separately into the inlet of the separation capillary. Owing to their different electrophoretic mobilities, spontaneous mixing of the two plugs takes place at the initial stage of electrophoresis, allowing the analytes to be derivatized on-column, prior to normal CE separation and detection. Such an arrangement eliminates most of the drawbacks of postcolumn detection mentioned above. However, it is only applicable to analytes with the appropriate functional groups. Clearly, NMCs are out of such a category. In the course of our study, we found that quaternary ammonium salts with a long alkyl chain were capable of catalyzing the degradation of NMCs with a significant reduction in the required alkaline concentration as well as reaction temperature. Since this type of quaternary ammonium salts can also act as micelle-forming surfactants in MEKC, we were prompted to create a quaternary ammonium salt-mediated MEKC system and explore the possibility of using a continuous capillary directly as the chambers for thermal decomposition and fluorescence detection. In this paper, the fundamental characteristic of the quaternary ammonium salt-assisted decomposition of NMCs is investigated first. Following that, a cetyltrimethylammonium bromide (CTAB)mediated MEKC separation system allowing on-column decomposition and derivatization is described, together with the instru(23) Bardelmeijer H. A.; Lingeman H.; de Ruiter C.; Underberg, W. J. M. J. Chromatogr., A 1998, 807, 3-26. (24) Zhu, R.; Kok, W. Th. J. Pharm. Biomed. Anal. 1998, 17, 985-999. (25) Rose, D. J.; Jorgenson, J. W. J. Chromatogr. 1988, 447, 117-131. (26) Cheng, Y. F.; Wu, S. L.; Chen D. Y.; Dovichi N. J. Anal. Chem. 1990, 62, 496-503. (27) Alkin, M.; Weinberger, R.; Sapp, E.; Moring, S. Anal. Chem. 1991, 63, 417422. (28) Rose, D. J. J. Chromatogr. 1991, 540, 343-353. (29) Gilman, S. D.; Ewing, A. G. Anal. Chem. 1995, 67, 58-64.
mental setup. Finally, the fluorescence detection of 10 NMCs is presented, and the results are compared with that of UV detection. For the first time, we demonstrated that, with appropriate buffer formulation, an intact capillary can serve consecutively as chambers for separation, decomposition, derivatization, and detection. The chemistry and instrumentation involved may be of great utility for the determination of a large number of other chemicals with similar structural features. EXPERIMENTAL SECTION Reagents. N-Methylcarbamate standards were supplied by Chem Service Inc. (West Chester, PA). Other chemicals and solvents were common brands such as Sigma (St. Louis, MO) and Fluka (Buchs, Switzerland). Stock solutions of individual pesticides and standard mixture were prepared in methanol. The injection sample was freshly made by diluting the standard mixture with the running electrolyte. The running electrolyte was prepared by dissolving required amounts of CTAB, OPA, and 2-mercaptoethanol in appropriate borate buffer. All buffer solutions were filtered through 0.45-µm nylon filtration disks (Whatman, Clifton, NJ). Water was collected from a Barnstead Nanopure Ultrafiltration unit (Boston, MA). Apparatus. The CE system was laboratory-made, which consisted of a LS30-2R4-3/3.5 high-voltage power supply (High Voltage Technology Inc., Yonkers, NY), a modified RF-551 spectrofluorometric detector (Shimadzu Corp., Kyoto, Japan) with excitation and emission wavelength set at 340 and 450 nm, respectively, and a Shimadzu C-R6A integrator. The separation tube was a bare fused-silica capillary with i.d. 50 µm, o.d. 375 µm, and total length 90 cm. The fluorescence detection window was made at 77 cm from the capillary inlet. A new capillary was preconditioned as before.22 To enable on-column thermal decomposition of NMCs, a heating site was created at the capillary downstream, by prodding the capillary diametrically through the external circulating tube of a Thermomix U heating circulator (B. Braun, Melsungen, Germany). As the hot oil from the heating circulator was driven through the external circulating tube, the part of the capillary buried inside the circulating tube would be heated concurrently, hence producing a narrow heating zone along the capillary, the temperature of which could be controlled conveniently and precisely via the heating circulator. To prevent leakage of oil following the burial of the capillary into the circulating tube, the tube itself must be thick walled and of good resilience. The type of tubing (0.64 cm i.d., 0.48 cm wall thickness) used in a vacuum system was found to be most suitable for such a purpose. To embed the separation capillary into the tubing, a 0.7-mm syringe needle was employed to prod across the tubing wall. Then, the capillary was inserted into the needle. Finally, the syringe needle was pulled out of the tubing, leaving the capillary buried firmly inside the tubing. In this way, the heating site could be positioned flexibly at any part of the capillary. For the present study, it was located at 65 cm from the inlet of the capillary. Figure 1 illustrates the schematic layout of the whole assembly. From the zoom view in Figure 1, it can be seen that the total length of the hot part of the capillary was roughly equivalent to the outer diameter of the circulating tube (∼1.6 cm). The unheated areas of the capillary were left as in usual separations, with no additional insulation or cooling.
Figure 1. Schematic presentation of the capillary layout. An expanded view of the cross section of the heating site is shown in the lower section.
Safety Consideration. It is necessary to exert reasonable caution when the high voltage is on. Some of the NMCs, such as aldicarb and carbofuran are highly toxic; thus care must be taken when these chemicals are handled. If necessary, these compounds can be decomposed with concentrated sodium hydroxide solution. RESULTS AND DISCUSSION To avoid the great difficulty in creating two microreactors along the capillary body as described in the introductory section, our initial investigation of the possible fluorescence detection of NMCs in the MEKC format had been focused on the introduction of alkaline and derivatizing reagents simultaneously via a single gap reactor. Such an attempt was proven to be unsuccessful, due mainly to the fact that, to effect a rapid decomposition of NMCs under an ambient temperature, a very high concentration of alkaline (>1 M) had to be applied through the gap. Owing to the high conductivity of the concentrated alkaline solution,, the analytes, together with the liberated methylamine and the generated fluorescent product, would all experience a very low electric field at and after the gap junction, hence causing considerable band broadening and prohibitively long migration times.30 Therefore, it was imperative to seek appropriate chemistry to bring the decomposition pH down to a level acceptable to the common CE separation. Following intensive experiments, we discovered that adding quaternary ammonium salts into borate buffer (pH 9∼10) accelerated dramatically the thermal degradation process of NMCs. Table 1 compares the effect of different reaction media on the thermal decomposition yield of NMC, using aldicarb as a model compound. In choosing the reaction solutions, we kept in mind that it would be of particular advantage if MEKC separation and thermal decomposition could be accomplished in a common medium. Therefore, three reaction solutions, each containing 10 mM borate buffer and 40 mM different surfactants that gave ideal performance in the prior MEKC separation of the model compounds with UV detection, were tested (Table 1) for their effects on the decomposition of NMCs. Table 1 shows clearly that the two solutions containing CTAB and myristyltrimethylammonium bromide (MTAB) promoted strongly the decomposition reaction, whereas the borate buffer alone or borate buffer with the anionic surfactant sodium dodecyl sulfate (SDS) showed no sign of such an effect. Meanwhile, the reaction yields for the two solutions containing quaternary ammonium salts were very close to that for 0.1 M sodium (30) Kuhr, W. G.; Licklider, L.; Amankwa, L. Anal. Chem. 1993, 65, 277-282.
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Table 1. Comparison of Thermal Decomposition of Aldicarb in Different Mediaa reaction medium
rel yieldb (%)
10 mM borate buffer, pH 10 100 mM sodium hydroxide 40 mM CTAB-10 mM borate, pH 10
8 100c 100
40 mM MTAB-10 mM borate, pH 10
95
40 mM SDS-10 mM borate, pH 10
10
remarks
slight decomposition of CTAB detected moderate decomposition of MTAB detected
a
To perform the thermal decomposition, a sealed polypropylene vial filled with 10 µg/mL aldicarb in the respective medium was immersed into 95 °C water bath for 30 s. b To determine the yield of thermal decomposition, the reaction solution was cooled and then 0.5% OPA and 0.5% 2-mercaptoethanol were added. After 1 min, the fluorescence product was measured with a spectrofluorophotometer (λEX ) 340 nm, λEM ) 450 nm). The relative yield of thermal decomposition was expressed in terms of the relative fluorescence intensity measured. cTo ensure thorough decomposition of aldicarb in sodium hydroxide solution, the sample was heated in 95 °C water bath for 3 min instead of 30 s. The subsequent fluorescence measurement was conducted as given in (b).
hydroxide solution, which meant that the quaternary ammonium salt-assisted thermal decomposition of aldicarb could complete quantitatively within 30 s. To some extent, this quaternary ammonium salt-induced degradation of NMCs came about not as a surprise. It has long been known that the hydrolysis of NMCs follows a two-step mechanism as described below: base
ROC(O)NHCH3 9 8 ROH + CH3NdCdO slow fast
CH3NdCdO + H2O 98 CH3NH2 + CO2
(1) (2)
The first step, cleavage of the ester bond, is the rate-controlling step. It can be catalyzed by a base. Quaternary ammonium salts are common base catalysts in many organic reactions. In fact, Nondek et al. had used a strong anion-exchange resin (Aminex A-27) as the heterogeneous catalyst for thermal degradation of NMCs, in which the surface of the anion exchanger was claimed to be bonded with tetraalkylammonium groups.20 Comparing the two quaternary ammonium salts investigated, CTAB appeared to be the preferred one, mainly because it generated less fluorescence background than MTAB. The production of a fluorescence background seemed to result from the decomposition of a small proportion of the quaternary ammonium salt at the elevated temperature. The fluorescence background was formed upon the conjugation of released alkylamine with the derivatizing agents. The detailed mechanism for this side reaction of quaternary ammonium salts is so far unknown. However, a similar phenomenon had been reported previously by Nondek et al.20 The above findings suggested that CTAB-containing MEKC medium offered a key to our problem: it could effect not only the MEKC separation but also the subsequent thermal decomposition of NMCs. As such, the difficulty arising from the need for a high concentration of alkali at the gap reactor could be readily circumvented, since the thermal decomposition could now be carried out at pH 9-10, which was amenable to a typical MEKC system. More importantly, with such a running buffer, on-column thermal decomposition of NMCs could now be performed, by 1444
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simply creating a hot zone at the downstream of the separation capillary. This made the gap reactor for introducing alkaline solution unnecessary. Up to here, if a conventional postcolumn derivatization strategy was to be followed, an interfacing structure such as a gap reactor would still required to introduce the derivatizing agents into the separation capillary. As stated above, since a heating device had to be mounted onto the separation capillary, it would be rather difficult to create a gap reactor nearby. To further simplify the equipment design, in situ derivatization was pursued, by incorporating the derivatizing components directly into the running buffer. With the adoption of such a strategy, the whole procedure of separation, thermal decomposition, derivatization, and fluorescence detection could be accomplished in a continuous capillary with complete elimination of any interfacing structure. To render a running buffer with in situ derivatization capability, the concentrations of the derivatizing components in the running buffer must be optimized in the first place. During this process, the following criteria should be kept in mind: (1) The concentrations of derivatizing agents must be held as low as possible so as not to interfere with the prior MEKC separation; (2) the highest sample fluorescence should be sought; (3) the presence of the derivatizing components in the running buffer should not pose any negative effect on the thermal decomposition of NMCs. In tagging free primary amines, it had been known that the concentrations of OPA and 2-mercaptoethanol, once exceeding certain thresholds, would have no effect on the ultimate fluorescence yields. 31 However, in the present reaction system, the influences of OPA and 2-mercaptoethanol on the thermal decomposition of NMCs as well as the background fluorescence intensity had to be taken into consideration. Figure 2 shows the effects of concentrations of OPA and 2-mercaptoethanol in the reaction medium on the thermal decomposition yields of NMCs. The study was conducted in a static manner, using carbofuran (5 ppm) as a model compound. Figure 2A depicts the influence of concentration of OPA on the fluorescence intensity of carbofuran, with concentration of 2-mercaptoethanol kept constant at 200 ppm. It was shown that the initial increase in the concentration of OPA was accompanied by a sharp increase in the net fluorescence intensity. After reaching ∼28 ppm (corresponding to a 10 times excess relative to the amount of carbofuran), further increases in OPA concentration did not bring further increase in fluorescence intensity. Such a trend was consistent with the situation in derivatizing a free amine. This meant that the existence of OPA in the reaction solution did not pose any effect on the thermal decomposition of NMCs. As for the background fluorescence, Figure 2A shows that it exhibited a pattern similar to that for carbofuran; i.e., following an initial rise in the background fluorescence with increases in the concentration of OPA, the background then became flattened. This was because the concentration of CTAB, which played the dominant role in the formation of the background fluorescence, was kept constant throughout the experiment. We noted that the background fluorescence contributed by OPA was negligible compared with that from CTAB. In contrast to OPA, the concentration of 2-mercaptoethanol imposed a significant impact on the yields of the thermal (31) Roth, M. Anal. Chem. 1971, 43, 880-82.
Figure 2. Effects of varying concentrations of OPA (A) and 2-mercaptoethanol (B) in the reaction medium on the fluorescence intensity of carbofuran (5 ppm). Reaction media: (A) 10 mM borate (pH 10) containing 40 mM CTAB and 100 ppm 2-mercaptoethanol, with varied concentrations for OPA. (B) 10 mM borate (pH 10) containing 40 mM CTAB and 28 ppm OPA, with varied concentrations for 2-mercaptoethanol. The test was conducted in a static manner, for which the sample was dissolved in the corresponding medium and then reacted at 95 °C for 30 s. The fluorescence intensity of carbofuran was corrected by subtraction of the background fluorescence contributed by CTAB.
decomposition of both NMCs and CTAB, as shown in Figure 2B. It can be seen that, when the concentration of 2-mercaptoethanol was varied between 5 and 200 ppm, there was rather stable net fluorescence intensity for carbofuran. However, further increase in its concentration led to a gradual decrease in the fluorescence yield. When the concentration of 2-mercaptoethanol reached 2000 ppm, the net fluorescence intensity of carbofuran dropped to near zero. This phenomenon suggests that the thermal decomposition of NMCs was inhibited by a high concentration of 2-mercaptoethanol. This “poisoning” effect of 2-mercaptoethanol on the thermal decomposition of NMCs was presumably due to the formation of ion pairs between the quaternary ammonium ions and thiolate anions (pKa ≈ 10 for thiol), as illustrated below: Note that the thermal decomposition of NMCs was catalyzed by CTAB or, more likely, by its base form, cetyltrimethylammonium hydroxide (CTAH). When a high concentration of 2-mercaptoethanol was introduced into the catalytic system, a substantial proportion of the quaternary ammonium ions would be “trapped” by 2-mercaptoethanol to form the corresponding ion pairs, leaving smaller amount of “free” cetyltrimthylammonium ions for participation in the catalytic process. Consequently, slower thermal decomposition of NMCs was observed. This was a probable scenario, because 2-mercaptoethanol was believed to have high
affinity toward quaternary ammonium ions, considering its alkyl backbone and the polarizability of the thiolate anions.32 Nevertheless, the presence of a relatively high concentration of 2-mercaptoethanol, on the other hand, had its merit. This can be seen in Figure 2B, from the line reflecting the background fluorescence that originated from CTAB. It shows that a higher concentration of 2-mercaptoethanol in the reaction medium resulted in a lower fluorescence background. Such a phenomenon might hint that the thermal stability of the quaternary ammonium ions could be strengthened by the ion-pairing mechanism. From the point of view of detecting low concentrations of NMCs, lower background fluorescence was desired, which meant that a sufficiently high concentration of 2-mercaptoethanol would be beneficial. Taking the above two aspects into account, the appropriate concentration for 2-mercaptoethanol was found to be 200 ppm (see Figure 2B). It should be noted that, in our case, the concentration of 2-mercaptoethanol in the catalytic system was rationalized on the basis of stepwise assays. The exact optimal concentration of 2-mercaptoethanol may be located with a systematic approach such as sequential simplex optimization. Following the optimization of the composition of the running medium, the thermal decomposition temperature became the most critical parameter to be determined. To locate the optimal temperature under which all the investigated NMCs underwent quantitative decomposition, the 10 model compounds were tested at different temperatures using the optimized reaction medium, in an off-line manner. And similarly, the corrected fluorescence intensity was employed as a gauge for the progress of the thermal decomposition. We did not attempt to monitor the reaction kinetics of the thermal decomposition of every NMC. However, on the basis of our observation of the thermal decomposition of aldicarb and carbofuran, the reaction yields were essentially constant when these two NMCs were heated under the optimum conditions for a period ranging between 15 s and 1 min. To facilitate a direct comparison of the experimental results, all the thermal decomposition experiments were performed at duration of 30 s. As will be discussed later, the 30-s duration was a close approximation of the time in which NMCs traveled through the heating zone in the real separation system. From the experimental results presented in Figure 3, it is found that, although the individual N-methylcarbamates exhibited their own critical temperatures at which a substantial breakdown occurred, quantitative decomposition of all NMCs could be achieved at 100 °C. It is worth noting that, at such temperature, (32) Weiss, J. Ion Chromatography, 2nd ed.; VCH: Weiheim, 1995; Chapter 3.
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Figure 3. Influence of reaction temperature on the decomposition yields of 10 NMCs. The test was carried out in a static manner, as in Figure 2. Reaction medium: 10 mM borate (pH 9.0) containing 40 mM CTAB, 28 ppm OPA, and 200 ppm 2-mercaptoethanol; reaction time 30 s. The corrected relative fluorescence intensity was obtained by comparing the net fluorescence intensity of a NMC with respect to that with complete decomposition of the respective NMC.
the thermal decomposition could be carried out without concern about boiling of the reaction medium, because the actual boiling point of the electrophoretic medium, in the presence of the buffer salt and CTAB surfactant, would be higher than 100 °C. In the meantime, it was interesting that, in this particular catalyzing system, the oxime-type NMCs such as aldicarb, methomyl, and oxamyl possessed breakdown temperatures similar to those of the phenoxyl-type NMCs, whereas on a catalytic bed the former required much higher temperatures than the latter.18,20 This might signify that, as far as promoting the thermal hydrolysis of NMCs was concerned, the present homogeneous catalyzing system worked more effectively than the heterogeneous catalytic bed. After the optimization of the separation medium and thermal decomposition temperature, the feasibility of on-column fluorescence detection of NMCs was finally evaluated with the instrumentation as described in Figure 1. The separation capillary and the buffer reservoirs were all filled with the optimum running medium, consisting of 10 mM borate buffer, 40 mM CTAB, 28 ppm OPA, and 200 ppm 2-mercaptoethanol. Upon the application of a negative high voltage (electroosmotic flow inside the capillary was reversed becuase of CTAB), normal MEKC separation took place within the “cold” part of the capillary stretching from the capillary inlet to the position immediately before the heating site. Thermal decomposition of the separated NMCs then occurred in the heated zone. The methylamine thus released experienced a rapid in situ derivatization. Finally, the fluorescent adduct was transported to the capillary downstream to be detected. In our experimental design, the heating zone was confined within a narrow region (∼1.6 cm) of the capillary. This enabled the creation of a sharp temperature pulse at the reaction chamber, and consequently, zone dispersion was contained at an acceptable level. Overall, the whole system was operated similarly to a conventional MEKC separation with fluorescence detection, except that 1446
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the temperature at the heating zone needed to be manipulated properly to enable thorough thermal decomposition of the resolved NMCs. The rise in temperature at the thermal decomposition zone was the result of the heat exchange between the circulating fluid and the electrophoresis solution via the capillary wall. Thus, the equilibrium temperature of the solution inside the heating zone was dictated by the moving speed of the solution inside the capillary, or more specifically, the velocity of the electroosmotic flow (EOF), as well as the temperature of the hot fluid from the heating circulator. In principle, the magnitude of EOF could be adjusted by manipulating the applied voltage and buffer composition (ionic strength, pH, CTAB concentration, etc.). Unfortunately, these adjustments would invoke concurrent changes in the migration times or sometimes even the separation selectivities for NMCs. On the contrary, adjusting the temperature setting on the heating circulator posed practically no disturbance on the advance MEKC separation. Thus, the manipulation of reaction temperature was made through the heating circulator. It was found that, with a stepwise increase in the temperature of the heating fluid from 90 to 120 °C, the peak heights of NMCs rose rapidly. However, when the heating temperature reached 130 °C, partial overlapping of neighboring peaks occurred. This was somewhat expected, as an excessive heating at the reaction zone would give rise to larger band broadening. In view of the above problem, the heating temperature was kept at 120 °C during our experiments. It should be noted that this temperature was higher than that for the off-line static study. This was understandable because, during the dynamic separation, to make the actual temperature of the flowing solution approach 100 °C, the temperature of the heating source had to be a bit higher, due to the heat-exchange process. A typical electropherogram for the fluorescence detection of the 10 NMCs at a concentration of 5 ppm is presented in Figure 4. It shows that satisfactory separation of all NMCs was achieved despite the temperature inconsistency along the separation capillary. The pH value of the running buffer in this case was chosen to be 9.0. As with pH 10, peak heights for carbofuran and promecarb were subdued, probably due to their partial hydrolysis in the course of MEKC separation. It should be pointed out that, for this particular separation system, the change in buffer pH would pose a stronger effect on thermal decomposition of NMCs than on separation of NMCs. Due to the electrical neutrality of NMCs, no significant alteration in separation selectivity was expected by changing the running buffer pH. In Figure 4, most NMCs exhibited quite similar fluorescence intensities, which was in agreement with the situation in the offline test (see Figure 3). Such a result suggested that the anlytes had experienced essentially the same thermal decomposition condition as in the off-line study. While the reaction temperature could be controlled directly via the heating circulator, the factors affecting the times of the dynamic thermal decomposition of individual NMCs were more complicated. On the basis of Figure 4 and the capillary layout as described in the Experimental Section, it could be estimated that the time of separated NMCs in contact with the heating zone was in the range of 15∼30 s, which was indeed comparable to the duration used for the static tests. A major exception in Figure 4 is that methamyl (peak 2) exhibited relatively low response as compared with the other NMCs. Retrospective to Figure 3, it can be seen that methamyl required
introduce an even more significant baseline shift on the electropherogram. In this case, the background fluorescence, although it may appear merely as a “signal offset” (hence shall not directly affect the improvement in the ultimate signal-to-noise ratios of the analytes), is undesirable. Therefore, to achieve the best performance, a proper way to suppress the background fluorescence should be sought in concurrence to LIF detection. Perhaps the only limitation of this new detection method for NMCs is a moderate loss of column efficiency due to the thermal decomposition step. As indicated in Figure 4, the typical plate numbers for the model compounds were ∼50 000. Such efficiency is lower than that for an orthodox MEKC system with plate numbers typically better than 100 000, yet it is significantly higher than that achievable with conventional HPLC. FUTURE WORK Figure 4. Typical MEKC separation of 10 NMCs (5 ppm each) with on-column thermal decomposition and fluorescence detection. Running electrolyte: 10 mM borate-40 mM CTAB (pH 9.0) with 28 ppm OPA and 200 ppm 2-mercaptoethanol. Capillary: i.d. 50 µm, total length 90 cm. Heating site and fluorescence detection window were respectively located at 65 and 77 cm from the inlet of the capillary. Applied voltage, -20 kV. Temperature for the heating circulator, 120 °C. Fluorescence detection: λEX ) 340 nm, λEM ) 450 nm. Peak identifications: 1, oxamyl; 2, methamyl; 3, aldicarb; 4, propoxur; 5, carbofuran; 6, aminocarb; 7, isoprocarb; 8, trimethacarb; 9, fenobucarb; 10, promecarb.
a relatively higher temperature to be decomposed thoroughly. Thus, a possible cause for the low peak height of methamyl in the dynamic separation was that the actual temperature at the heating site might have been slightly below 100 °C. A prominent feature of the above electropherogram is the existence of a baseline climb at approximately the fourth minute after injection. Again, this was accredited to the decomposition of a small proportion of CTAB at the elevated temperature with the release of alkylamine, which quickly reacted with OPA/2mercaptoethanol to give the corresponding fluorogenic compound. Since this process persisted when the system was in operation, a significant but relatively stable fluorescence background was generated. This fluorescence detection scheme proved to be valid in meeting our original goal, that is, to enhance the detection sensitivity of NMCs in a MEKC format. The detection limits based on a signal-to-noise ratio of 3 were found to be around 0.5 ppm for all NMCs. Such detection sensitivity is at least 5 times better than that in UV detection. Note that the present result had been based on a very simple design of a detection cell, in which the capillary was placed directly in the excitation light path without any focusing elements. Further improvement in the detection limits is possible if refinements of the radiation and collection optics are made. An even more dramatic improvement of detection limits can be anticipated if laser-induced fluorescence detection is employed. This can be achieved by using a He-Cd laser operated at 325 nm or replacing OPA with naphthalene-2,3dicarboxyaldehyde (NDA) and operating the He-Cd laser at 442 nm. It can be expected that, by incorporating LIF detection into the current system, the background fluorescence caused by decomposition of CTAB may increase accordingly, which would
It is believed that, in conjunction with a suitable CE separation format, e.g., MEKC or capillary electrochromatography (CEC), this fluorescence detection scheme might be applicable in the determination of many other compounds. In principle, using the tactics as described in this study, any molecules containing amide moieties may be hydrolyzed with the release of primary amines or ammonia, which can be subsequently converted to the corresponding fluorogenic derivatives and finally be detected with a fluorescence detector. Amido groups can be found in many categories of pharmaceuticals and agrochemicals. By adapting similar buffer chemistry, new detection methods could be created. Further work is in progress in our laboratory to explore the applicability of this new detection scheme on a more general basis. CONCLUSION Fluorescence detection of NMCs has been realized in a MEKC separation with potential advantages of significant improvements in detection sensitivity and selectivity. This new detection scheme hinges on CTAB-catalyzed thermal degradation of NMCs. By incorporating OPA/2-mercaptoethanol into the CTAB-mediated MEKC separation buffer, together with the creation of a welllocalized heating zone on the downstream of the separation capillary, we have succeeded in utilizing an intact capillary as chambers for separation, thermal decomposition, derivatization, and detection. Due to this unique approach, on-column thermal decomposition and derivatization have been achieved without relying on any difficult “microsurgery” on the capillary body that is often required for postcolumn derivatization in CE, hence resulting in a very simple yet effective operation system. The strategy used here could be applied for the fluorescence determination of numerous compounds with similar structural features. ACKNOWLEDGMENT The authors thank the National University of Singapore for providing financial support to this research.
Received for review August 17, 1999. Accepted December 30, 1999. AC990928D Analytical Chemistry, Vol. 72, No. 7, April 1, 2000
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