A fluorescence detection scheme for capillary electrophoresis of N- methylcarbamates with on-column thermal decomposition and derivatization

Article abstract of DOI:10.1021/ac990928d

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.

Full text of DOI:10.1021/ac990928d

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
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 vaporiza-
tion.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 RP-
HPLC is its compatibility with aqueous samples, which allows on-
column 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 inad-
equate 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
This paper describes a fluorescence detection method for
N-methylcarbamate (NMC) pesticides in micellar electro-
kinetic chromatography (MEKC) separation. Fulfillment
of the fluorescence detection hinged on the discovery that
quaternary ammonium surfactants (particularly cetyltri-
methylammonium 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 com-
ponents (o-phthaldialdehyde/ 2-mercaptoethanol) was for-
mulated, 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 1 0 NMC
compounds was achieved, with column efficiencies typi-
cally higher than 5 0 0 0 0 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 HP LC. 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.
(1) McGarvey, B. D. J. Chromatogr. 1 9 9 3 , 642, 89-105.
(2) Liska, I.; Slobodnik, J. J. Chromatogr., A 1 9 9 6 , 733, 235-258.
(3) Yang, S. S.; Goldsmith, A. I.; Smetena I. J. Chromatogr., A 1 9 9 6 , 754, 3-16.
(4) Fishbein, L.; Zielinski, W. J. Chromatogr. 1 9 6 5 , 20, 9-14.
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150.
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59, 188-196.
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12, 40-45.
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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 © 2000 American Chemical Society
Published on Web 02/23/2000
Analytical Chemistry, Vol. 72, No. 7, April 1, 2000 1441

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 (hy-
droxyethylthio-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, RP-
HPLC 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 regula-
tion 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 deter-
mination 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 perfor-
mance, 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 require-
ment 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 on-
column stacking, which contributed to sample enrichment of
several thousandfold, was required.22
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 microfab-
ricaiton 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/ fluores-
cence 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 detec-
tion. 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 am-
monium 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 decom-
position and derivatization is described, together with the instru-
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
(23) Bardelmeijer H. A.; Lingeman H.; de Ruiter C.; Underberg, W. J. M. J.
Chromatogr., A 1 9 9 8 , 807, 3-26.
(24) Zhu, R.; Kok, W. Th. J. Pharm. Biomed. Anal. 1 9 9 8 , 17, 985-999.
(25) Rose, D. J.; Jorgenson, J. W. J. Chromatogr. 1 9 8 8 , 447, 117-131.
(26) Cheng, Y. F.; Wu, S. L.; Chen D. Y.; Dovichi N. J. Anal. Chem. 1 9 9 0 , 62,
496-503.
(19) Moye, H. A.; Scherer, S. J.; John, P. A. Anal. Lett. 1 9 7 7 , 1049-1054.
(20) Nondek, L.; Frei, R. W.; Brinkman, U. A. Th. J. Chromatogr. 1 9 8 3 , 282,
141-150.
(27) Alkin, M.; Weinberger, R.; Sapp, E.; Moring, S. Anal. Chem. 1 9 9 1 , 63, 417-
422.
(21) Wu, Y. S.; Lee, H. K.; Li, S. F. Y. J. Microcolumn Sep. 1 9 9 8 , 10, 239-247.
(22) Wu, Y. S.; Lee, H. K.; Li, S. F. Y. J. Microcolumn Sep. 1 9 9 8 , 10, 529-535.
(28) Rose, D. J. J. Chromatogr. 1 9 9 1 , 540, 343-353.
(29) Gilman, S. D.; Ewing, A. G. Anal. Chem. 1 9 9 5 , 67, 58-64.
1442 Analytical Chemistry, Vol. 72, No. 7, April 1, 2000

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 cham-
bers 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
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.
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-mercapto-
ethanol 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 Ultrafiltra-
tion unit (Boston, MA).
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 main-
ly 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 fluor-
escent product, would all experience a very low electric field at
and after the gap junction, hence causing considerable band broad-
ening and prohibitively long migration times.30 Therefore, it was
imperative to seek appropriate chemistry to bring the decomposi-
tion 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) acceler-
ated 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 com-
pound. 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 com-
pounds with UV detection, were tested (Table 1) for their effects
on the decomposition of NMCs.
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 pre-
cisely 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 cir-
culating tube (∼1.6 cm). The unheated areas of the capillary were
left as in usual separations, with no additional insulation or cooling.
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. 1 9 9 3 , 65, 277-282.
Analytical Chemistry, Vol. 72, No. 7, April 1, 2000 1443

simply creating a hot zone at the downstream of the separation
capillary. This made the gap reactor for introducing alkaline
solution unnecessary.
Table 1. Comparison of Thermal Decomposition of
Aldicarb in Different Mediaa
rel yieldb
(%)
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 incor-
porating the derivatizing components directly into the running
buffer. With the adoption of such a strategy, the whole procedure
of separation, thermal decomposition, derivatization, and fluores-
cence detection could be accomplished in a continuous capillary
with complete elimination of any interfacing structure.
reaction medium
remarks
10 mM borate buffer, pH 10
100 mM sodium hydroxide
8
100c
100
40 mM CTAB-10 mM borate, pH 10
slight decomposition of
CTAB detected
moderate decomposition
of MTAB detected
40 mM MTAB-10 mM borate, pH 10
95
10
40 mM SDS-10 mM borate, pH 10
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. To determine the yield of
b
thermal decomposition, the reaction solution was cooled and then 0.5%
OPA and 0.5% 2-mercaptoethanol were added. After 1 min, the fluor-
escence product was measured with a spectrofluorophotometer (λEX
) 340 nm, λEM ) 450 nm). The relative yield of thermal decomposi-
tion was expressed in terms of the relative fluorescence intensity
c
measured. To 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).
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 concentra-
tions 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
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:
certain thresholds, would have no effect on the ultimate fluores-
31
cence yields.
However, in the present reaction system, the
ROC(O)NHCH3 base8 ROH + CH3NdCdO
(1)
(2)
influences of OPA and 2-mercaptoethanol on the thermal decom-
position of NMCs as well as the background fluorescence intensity
had to be taken into consideration.
slow
CH3NdCdO + H2O fast8 CH3NH2 + CO2
Figure 2 shows the effects of concentrations of OPA and
2-mercaptoethanol in the reaction medium on the thermal de-
composition 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 fluores-
cence intensity of carbofuran, with concentration of 2-mercapto-
ethanol 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.
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 gen-
erated less fluorescence background than MTAB. The production
of a fluorescence background seemed to result from the decom-
position 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 deriv-
atizing 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 decom-
position 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
In contrast to OPA, the concentration of 2-mercaptoethanol
imposed a significant impact on the yields of the thermal
(31) Roth, M. Anal. Chem. 1 9 7 1 , 43, 880-82.
1444 Analytical Chemistry, Vol. 72, No. 7, April 1, 2000

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 suf-
ficiently 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.
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 fluores-
cence contributed by CTAB.
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 decom-
position 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.
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-mercapto-
ethanol. 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, cetyltrimethylammo-
nium hydroxide (CTAH). When a high concentration of 2-mer-
captoethanol 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 partici-
pation 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
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.
Analytical Chemistry, Vol. 72, No. 7, April 1, 2000 1445

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 decom-
position 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 selectivi-
ties 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 capil-
lary. 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.
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 fluores-
cence detection of NMCs was finally evaluated with the instru-
mentation 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.
In Figure 4, most NMCs exhibited quite similar fluorescence
intensities, which was in agreement with the situation in the off-
line 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
Overall, the whole system was operated similarly to a conven-
tional MEKC separation with fluorescence detection, except that
1446 Analytical Chemistry, Vol. 72, No. 7, April 1, 2000

introduce an even more significant baseline shift on the electro-
pherogram. 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 perfor-
mance, 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. Run-
ning 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, fenobu-
carb; 10, promecarb.
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 corre-
sponding 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.
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/ 2-
mercaptoethanol 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-2,3-
dicarboxyaldehyde (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
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 well-
localized 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 deter-
mination 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 1447