Sensitized and Quenched Phosphorescence as a Detection Mode in

The potential of sensitized and quenched phosphorescence of biacetyl as an optical detection mode in capillary electrophoresis (CE), complementary to ...
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Anal. Chem. 1999, 71, 1384-1390

Sensitized and Quenched Phosphorescence as a Detection Mode in Capillary Electrophoresis Jacobus Kuijt, Udo A. Th. Brinkman, and Cees Gooijer*

Department of Analytical Chemistry and Applied Spectroscopy, Free University, de Boelelaan 1083, NL-1081 HV Amsterdam, The Netherlands

The potential of sensitized and quenched phosphorescence of biacetyl as an optical detection mode in capillary electrophoresis (CE), complementary to absorption and fluorescence detection, was explored. From 24 naphthalenesulfonates (NS) that were studied in batch experiments, 5 NS were used as test compounds in CE. The technique is based on the intense phosphorescence emission of biacetyl (present as a constituent of the CE buffer) at room temperature in deoxygenated liquid solutions. A simple device, based on purging with nitrogen gas, was developed to meet this deoxygenation requirement in CE. A standard liquid chromatography luminescence detector, provided with a pulsed xenon light source, was used for detection. In view of the phosphoresence lifetime of biacetyl (70 µs under present solution conditions), the background caused by scattered excitation light could be readily suppressed by using a delay time for detection. Both phosphorescence modes can be applied at a 0.02 M biacetyl concentration, though in the quenched mode a biacetyl concentration of 0.05 M yields better results. From the five test analytes considered, three show sensitized phosphorescence and two dynamically quenched phosphorescence. Though various experimental parameters still have to be optimized further, the results are quite encouraging: under stacking conditions (pt ) 768 mbar‚s), detection limits ranged from 5 × 10-8 to 4 × 10-7 M. Optical Detection Methods in Capillary Electrophoresis. Over the last five years capillary electrophoresis (CE) has gained much in popularity, mainly because of the high resolutions that are attainable in the separation of both ionogenic compounds and neutral molecules. Unfortunately, the analyte detectability achievable in CE is rather poor when optical detection methods are used.1 This, until now, has largely inhibited the use of CE in environmental analysis. Important causes for the low sensitivity are the short path length through the capillary, the small illuminated surface, and the short maximum detector time constants that can be used. In addition, optical detection methods suffer from inadequate coupling of light to the liquid core of the (1) Yeung, E. S. In Advances in Chromatography; Brown, P. R., Grushka, E., Eds.; Marcel Dekker: New York, 1995; Vol. 35, Chapter 1.

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capillary, which results in a low photon flux in the capillary and leads to high noise levels. Furthermore, the illumination of the capillary wall can lead to an excessive production of stray light.1 These problems can be solved partly by using ball lenses and narrow slits.1,2 With ultraviolet (UV) absorption detection, which is still used most often in CE, concentration detection limits in the 10-6-10-7 M range can only be expected in the most favorable cases,3 i.e., with high analyte extinction coefficients and the use of analyte preconcentration by stacking. This explains why, currently, much effort is devoted to improving concentration detection limits in CE by a combination of appropriate sample handling for trace enrichment and cleanup and the use of alternative optical detection schemes. These detection methods should not only be more sensitive but, maybe even more important, should also provide additional selectivity to fully exploit the separation potential of CE. In view of the extremely small detector cell volumes one has to deal with in CE, in the literature much attention has been devoted to the development of laser-based detection techniques,4-7 although the use of chemiluminescence has been studied also.1 Among these techniques, laser-induced fluorescence (LIF) is by far superior with regard to detection limits. Unfortunately it has a limited application range, unless chemical derivatization is included. In addition, with analytes exhibiting native fluorescence, expensive lasers emitting in the deep UV are often required. In the present paper, a conventional luminescence detection method, room-temperature phosphorescence in the liquid phase (RTPL), is studied, which uses a conventional liquid chromatography (LC)-type luminescence detector. RTPL was developed in our laboratory for use with LC in the early 1980s,8,9 but no applications in CE have been reported in the literature so far. (2) Walbroehl, Y.; Jorgenson, J. W. J. Chromatogr. 1984, 315, 135-143. (3) Kok, S. J.; Koster, E. H. M.; Gooijer, C.; Velthorst, N. H.; Brinkman, U. A. Th.; Zerbinati, O. J. High Resolut. Chromatogr. 1996, 19, 99-104 (4) Yeung, E. S.; Wang, P.; Li, W.; Giese, R. W. J. Chromatogr. 1992, 608, 73-77. (5) Kok, S. J.; Kristenson, E. M.; Gooijer, C.; Velthorst, N. H.; Brinkman, U. A. Th. J. Chromatogr., A 1997, 771, 331-341. (6) Milofsky, R. E.; Malberg, M. G.; Smith, J. M. J. High Resolut. Chromatogr. 1994, 17, 731-732. (7) Milofsky, R.; Spaeth, S. Chromatographia 1996, 42, 12-16. (8) Donkerbroek, J. J.; Eikema-Hommes, N. J. R. van; Gooijer, C.; Velthorst, N. H.; Frei, R. W. Chromatographia 1982, 15, 218-222. (9) Donkerbroek, J. J.; Eikema-Hommes, N. J. R. van; Gooijer, C.; Velthorst, N. H.; Frei, R. W. J. Chromatogr. 1983, 255, 581-590. 10.1021/ac981130h CCC: $18.00

© 1999 American Chemical Society Published on Web 02/17/1999

Figure 1. Energy diagram for sensitized phosphorescence. Iabs, rate of light absorption by the donor and kDf , kDnf, and kDisc, rate constants for fluorescence, nonradiative deactivation, and intersystem crossing from the donor S1 state, respectively. kDp , kDisc, and kDA t [A], rate constants for phosphorescence, intersystem crossing, and energy transfer to the acceptor T1 state, respectively. kAp , phosphorescence rate constant of the acceptor; kAnr, rate constant for nonradiative deactivation processes.

Two RTPL modes can be distinguished. In the sensitized mode the analyte, after being excited at its optimum excitation wavelength, transfers its excitation energy to biacetyl after which the phosphorescence of the latter compound is observed. In the quenched mode in which biacetyl is excited directly at 410 nm, the biacetyl phosphorescence quantum yield is reduced by a bimolecular quenching process involving the analyte molecules, which causes negative peaks to show up in the electropherogram. It should be noted that the quenched RTPL mode is fundamentally different from other indirect detection modes, such as, for example, indirect UV absorption detection, which are based on the displacement of chromophoric eluent (or electrophoretic buffer) constituents by the analytes.10,11 Sensitized and Quenched Phosphorescence. A simplified diagram for sensitized phosphorescence is shown in Figure 1. After excitation of the analyte, the lowest excited singlet state, S1, becomes populated. From this state, the lower-lying triplet state, T1, can be populated via intersystem crossing (ISC). Competing processes are fluorescence and nonradiative decay to the ground state. The triplet state of the analyte (donor) can be deactivated by phosphorescence, intramolecular radiationless decay, bimolecular quenching, or energy transfer to an acceptor molecule. Because of the high quenching rate constant (kq ∼ 1091010 M-1 s-1), bimolecular quenching of the T1 state by dissolved oxygen is normally the dominating deactivation path in liquids at room temperature, so that phosphorescence is not observed.12 Even in carefully deoxygenated solutions, the phosphorescence of most compounds is too slow (kp ∼ 10-1-10 s-1) to be of analytical value. Biacetyl, which has a fairly high kp value (kp ∼ 103 s-1), is a well-known exception. For this compound, intense phosphorescence emission can be easily observed in deoxygenated solutions. Sensitized phosphorescence detection can be used for analytes with a high intersystem crossing efficiency, ϑD ISC a large fraction of these molecules will reach the triplet state after excitation. (10) Yeung, E. S.; Kuhr, W. G. Anal. Chem. 1991, 63, 275A-282A. (11) Nielen, M. W. F. J. Chromatogr. 1992, 608, 85-92. (12) Donkerbroek, J. J.; Elzas, J. J.; Gooijer, C.; Frei, R. W.; Velthorst, N. H. Talanta 1981, 28, 717-723.

Interestingly, under favorable conditions, the intermolecular energy transfer to an acceptor molecule such as biacetyl can be a very rapid process (kt is typically 109 M-1 s-1 or higher), which is able to compete with bimolecular quenching of the analyte by oxygen in deoxygenated solutions. Therefore, intense sensitized phosphorescence of an appropriate acceptor molecule like biacetyl can be observed, provided that the triplet state of the donor molecule (analyte) is higher than the triplet state of the acceptor molecule.12 If inner filter effects are ignored (which applies at the low analyte concentrations considered), the intensity of sensitized phosphorescence can be written as12 D DA A Ip(sens) ) 2.303I0(λex)(λ [D]lϑD iscϑt ϑp ex)

(1)

In eq 1, I0(λex) is the intensity of the excitation light; the following three symbols represent the extinction coefficient of the donor, its concentration, and the optical path length; the last three symbols are the intersystem crossing efficiency of the donor (analyte), the efficiency of energy transfer to the acceptor (biacetyl), and the phosphorescence efficiency of the acceptor, respectively. The phosphorescence efficiency can be written as

ϑp ) kp/(kp + knp +

∑k [Q]) ) k τ q

p p

(2)

where kp is the phosphorescence emission rate constant, knp the rate constant for nonradiative decay, and ∑kq[Q] the sum of all effective unimolecular quenching rate constants (the products of bimolecular quenching rate constants and quencher concentrations). The resulting phosphorescence lifetime, which is actually observed, is given by τp. In the quenched phosphorescence mode, the phosphorophore (biacetyl) itself is excited and phosphorescence radiation with intensity I0 is emitted from its triplet state. Analytes with a lowerlying triplet state may then diminish the phosphorescence intensity from I0 to I; in other words, they can operate as bimolecular quenchers. The ratio I0/I is given by the SternVolmer equation

I0/I ) 1 + kq[Q]τ0

(3)

where kq, [Q], and τ0 are the quenching rate constant for quenching by the analyte, the analyte concentration, and the triplet lifetime of biacetyl in the absence of the quenching analyte, respectively. According to eq 3, small concentrations of quenchers (analytes) exhibit efficient quenching if τ0 is sufficiently high, i.e., higher than ∼0.1 ms, provided that the associated quenching rate constant is at its maximum value, i.e., if kq is diffusion- controlled. Batch experiments using biacetyl as an acceptor showed the favorable spectroscopic properties of this compound for sensitized RTPL.13 It has a very low extinction coefficient over the whole nonvacuum UV range, so that especially in CE where the optical path length is very short (typically 75 µm), relatively high biacetyl concentrations can be used without the problem of a high (13) Donkerbroek, J. J.; Gooijer, C.; Velthorst, N. H.; Frei, R. W. Anal. Chem. 1982, 54, 891-895.

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background caused by direct excitation. (As will be detailed below, direct excitation of biacetyl as needed in the quenched detection mode, can be done at 410 nm where the biacetyl absorption spectrum shows a maximum.) Moreover, because the biacetyl triplet state is at a rather low energy level (19 700 cm-1),13 quite a number of analytes will, in principle, be able to produce sensitized RTPL. Finally, biacetyl has a rather large phosphorescence rate constant, kp, which implies that the phosphorescence efficiency, ϑp, can be quite high. Within this context it should be noted that, in addition to RTPL, there is another mode of long-lived luminescence, i.e., Eu(III) and Tb(III) luminescence,14-19 applicable in both the sensitized14-18 and the quenched 19 modes. This mode of luminescence is hardly inhibited by the presence of oxygen in the solution, and no special devices to remove oxygen have to be used in LC. However, in sensitized lanthanide luminescence detection, this holds only for analytes that form stable complexes with Eu(III) and Tb(III) so that the energy transfer from the ligand to the metal ion is basically an intramolecular process, a condition that restricts its applicability as a detection method. Furthermore, there are only few analytes that show efficient dynamic quenching of lanthanide luminescence. Nevertheless, contrary to RTPL, some applications of lanthanide luminescence in CE6,7,14,15 have been reported. In this work, in which the emphasis is on nonmicellar capillary electrophoresis, both sensitized and quenched RTPL of biacetyl are used in a model separation of five naphthalenesulfonates (NS). The influence of the biacetyl concentration on the signal-to-noise (S/N) ratio is studied for both phosphorescence modes. An interesting feature is that the three detection modes (fluorescence, sensitized RTPL, quenched RTPL) can be used with the same instrumental setup and buffer composition (including the concentration of biacetyl): only a few detection parameters have to be changed. EXPERIMENTAL SECTION Chemicals. Demineralized and distilled water was used in all experiments. Biacetyl (97%) and 4-amino-5-hydroxy-2,7-naphthalenedisulfonic acid (96%) were purchased from Aldrich (Steinheim, Germany). 1-Naphthalenesulfonic acid (∼70%), 2,6-naphthalenedisulfonic acid (∼80%), 1,3,7-naphthalenetrisulfonic acid (∼70%), 4,5-dihydroxy-2,7-naphthalenedisulfonic acid, and other NS which were used in batch experiments only were kindly provided by Prof. O. Zerbinati (Turin, Italy). In the remainder of this text, the NS will be indicated by the positions of their functional groups, amino/hydroxy/sulfonic acid. For instance, 4-amino-5-hydroxy2,7-naphthalenedisulfonic acid will be denoted as 4/5/2,7. Substitution positions on the naphthalene molecule are numbered 1-8, starting at the most upper right position with the long side of the naphthalene molecule oriented along the horizontal axis. Boric acid (p.a.) and sodium hydroxide (Baker grade) were (14) Zhu, R.; Kok, W. Th. Anal. Chem. 1997, 69, 4010-4016. (15) Milofsky, R.; Bauer, E. J. High Resolut. Chromatogr. 1997, 20, 638-642. (16) Hirschy, L. M.; Dose, E. V.; Winefordner, J. D. Anal. Chim. Acta 1983, 147, 311-316. (17) DiBella, E. E.; Weissman, J. B.; Joseph, M. J.; Schultz, J. R.; Wenzel, Th. J. J. Chromatogr. 1985, 328, 101-109. (18) Mwalupindi, A. G.; Warner, I. M. Anal. Chim. Acta 1995, 306, 49-56. (19) Baumann, R. A.; Kamminga, D. A.; Derlagen, H.; Gooijer, C.; Velthorst, N. H.; Frei, R. W. J. Chromatogr. 1988, 439, 165-170.

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Figure 2. Instrumental setup for CE with sensitized and quenched phosphorescence detection. The setup consists of an ultrasonic bath and a nitrogen gas supply for deoxygenation, an HPLC pump for pumping the CE buffer and the sample (1 mL/min), a luminescence detector, and a CE system. Sample introduction in the CE capillary is achieved by hydrodynamic injection at a fixed time interval after switching the six-way valve. The 2-m PEEK restriction between pump and interface has been omitted for clarity. Inset: interface with the injection end of the capillary and the positive electrode inserted in it.

obtained from J. T. Baker (Deventer, The Netherlands) and were used to prepare the buffer solutions. All chemicals were used without further purification. Buffers were prepared freshly every day. Apparatus. An LS-50 luminescence spectrometer (PerkinElmer, Norwalk, CT) was used to record phosphorescence and fluorescence excitation and emission spectra. Samples were contained in a 1-cm quartz cuvette with a long glass setup, which could be closed with a stopper. Samples were deoxygenated with a gentle stream of nitrogen for ∼10 min before phosphorescence measurement. The instrumental setup for CE experiments is schematically drawn in Figure 2; it is composed of a homemade facility to remove oxygen from the CE buffer solution, a homemade interface to couple the buffer flow with the CE system following an approach developed in our laboratory,20 and the CE system including the luminescence detector. Oxygen was removed from 1 L of buffer solution by simultaneous purging with nitrogen gas and sonification in an ultrasonic bath. The deoxygenated buffer solution was pumped (flow rate, 1.00 mL/min) by an HPLC pump (LKB model 2150, Bromma, Sweden) to the homemade interface, which is described in the next section. To preclude oxygen from entering the buffer solution, all connections from the buffer reservoir to (20) Veraart, J. R.; Gooijer, C.; Lingeman, H.; Velthorst, N. H.; Brinkman, U. A. Th. Chromatographia 1997, 44, 581-588.

the interface were made of stainless steel tubing. The only exception was a 2-m flow restriction made of red PEEK tubing (i.d., 0.13 mm) positioned between the pump and the six-way injection valve to provide a pressure drop over the system. Automated injection with a 200-µL sample loop (Valco, Berkeley, CA) was achieved with a MUST HP 6 multiport stream switch (Spark Holland, Emmen, The Netherlands), triggered by the PrinCE CE injection/high-voltage system (Lauerlabs, Emmen, The Netherlands). All samples were filtered over 0.45-µm syringe filters (Schleicher & Schuell, Dassel, Germany) prior to injection. A fused-silica capillary (Composite Metals Service, Hallow, UK) with a total length of 108 cm was used (i.d., 75 µm; o.d., 375 µm). A detection window with a length of 4 mm was burned off at 56 cm from the inlet of the capillary. Fluorescence and phosphorescence signals were detected with an LS-40 luminescence LC detector (Perkin-Elmer, Beaconsfield, UK), in which the original LC detection cell had been replaced by a spacer, which was used to hold the capillary in place. A chart recorder (Kipp, Delft, The Netherlands) and a computer with Class-VP software (Shimadzu, Columbia, MD) were used for data collection. Interface. The interface (see inset in Figure 2) consisted of a 3.0-cm piece of gray PEEK tubing (i.d., 1.0 mm; volume, 24 µL) fixed in a 1/16-in. union (Valco) which was connected to the stainless steel tubing with finger tight to allow easy removal. The electrode in the interface functioning as the anode (+) was connected to ground, while the electrode in the outlet vial functioned as the cathode (-). Nitrogen gas was flushed around the interface. RESULTS AND DISCUSSION Batch Experiments. Sensitized phosphorescence excitation and emission spectra in demineralized and distilled water were recorded for the 24 NS studied by Kok et al.,5 using biacetyl (10-4 M) as an acceptor and the NS (5 × 10-6 M) as donor molecules. The nonsubstituted NS (with one, two, or three sulfonic acid groups) showed sensitized phosphorescence as depicted in Figure 3 for 0/0/2,6. As expected, the excitation spectrum is similar to the 0/0/2,6 fluorescence excitation spectrum, while the emission spectrum (λmax ) 513 nm) closely resembles the direct phosphorescence emission spectrum of biacetyl (performing excitation at 410 nm). For the hydroxy- and amino-substituted NS, no phosphorescence signals apart from the direct phosphorescence of biacetyl were observed. Apparently, these compounds are not able to sensitize biacetyl phosphorescence. This agrees well with the fact that the fluorescence spectra of the nonsubstituted NS show emission maximums in the 400-480-nm range, which suggests that their triplet states have a lower energy than the triplet state of biacetyl. Therefore no efficient energy transfer is possible. They might instead be efficient quenchers of biacetyl phosphorescence; if the triplet state energy for a particular analyte is ∼5 kcal/mol lower than that of biacetyl, the associated quenching process is diffusion-controlled and the bimolecular rate constant, kq, is at its maximum.12 Indeed, as will be outlined below, for the nonsubstituted NS that were used in the CE experiments (4/5/2,7; 0/4,5/ 2,7) we have measured high quenching rate constants. Capillary Electrophoresis with Phosphorescence Detection. For the CE experiments, five NS (0/0/1; 0/0/2,6; 0/0/1,3,7;

Figure 3. Spectra of 5 × 10-6 M 2,6-naphthalenedisulfonic acid in demineralized and distilled water obtained in batch experiments. (a) Fluorescence (1) and sensitized phosphorescence (2) excitation spectra, with emission wavelengths of 346 and 513 nm, respectively; (b) fluorescence emission spectrum; (c) sensitized phosphorescence emission spectrum (10-4 M biacetyl). The emission spectra were obtained with excitation at 230 nm. Table 1. Program for CE of Five NS step 1 2 3 4 5

pressure voltage (mbar) (kV) 0 -15 0 0 -180

0 0 0 -30 0

external event

duration (min)

contact closure on t ) 0.10 min contact opening on t ) 0.10 min

0.29 0.16 0.70 17.00 3.00

0/4,5/2,7; 4/5/2,7) were selected from the 24 studied in batch. CE was performed with aqueous 25 mM borate buffers (pH 8.5) which contained varying biacetyl concentrations. One liter of the buffer solution was simultaneously sonicated and purged with nitrogen for 1 h. The nitrogen gas flow was reduced and maintained at a low speed for the rest of the experiments. In this way, a stable baseline (λex ) 410 nm) was obtained within 2 h. With the CE program used in this work (see Table 1), it is possible to record more than 30 electropherograms with 1 L of CE buffer. Because biacetyl is a rather volatile compound, evaporation of biacetyl occurs due to the purging. Since, in the present setup, typically 10% of the biacetyl was lost from the solution during a working day, a fresh buffer solution was prepared daily. It was not necessary to deoxygenate the sample solutions prior to injection, because oxygen is separated from the migrating analyte zones during electrophoresis. For sensitized RTPL, the excitation and emission wavelengths were set to 230 and 513 nm, respectively. In the quenched RTPL mode, the emission wavelength was the same (513 nm) but excitation was at 410 nm. The excitation and emission slits were fixed at 10 nm. Electropherograms obtained with 0.02 M biacetyl in the buffer solution are presented in Figure 4, showing positive peaks for the three nonsubstituted NS and negative peaks for the two substituted ones (and for molecular oxygen), as expected. These, and electropherograms obtained with 0.005, 0.01, and 0.05 M biacetyl in the buffer Analytical Chemistry, Vol. 71, No. 7, April 1, 1999

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Figure 4. Electropherograms of five NS. Buffer: 25 mM borate, pH 8.5, containing 0.02 M biacetyl. Peaks: (1) oxygen; (2) 1-naphthalenesulfonic acid; (3) 4-amino-5-hydroxy-2,7-naphthalenedisulfonic acid; (4) 2,6-naphthalenedisulfonic acid; (5) 4,5-dihydroxy-2,7-naphthalenedisulfonic acid; (6) 1,3,7-naphthalenetrisulfonic acid. Detection: (a) sensitized RTPL; concentration of NS, 10-5 M; (b) quenched RTPL, concentration of NS, 2.5 × 10-6 M. All other parameters are given in the text.

solution, will be discussed in the sections addressing the sensitized and quenched modes, respectively. Oxygen Generation at the Anode. Owing to the (high) voltages applied during a CE separation, oxygen is produced at the anode and the question arises whether this oxygen generation inhibits RTPL detection. The following half-reaction applies:

(4OH- a 2H2O(l ) + O2(g) + 4e-) 2H2O(l) a 4H+ + O2(g) + 4eAs four electrons are involved in the formation of a single oxygen molecule, the concentration, [O2]el, generated during electrophoresis due to hydrolysis of water (overall reaction) follows from

[O2]el ) I/4Ff

(4)

where I is the measured electric current (A), F the Faraday constant (96 489 C/mol), and f the flow delivered by the pump expressed in liters per second. With the 25 mM borate buffer, a current of only ∼4 µA was produced. It follows from eq 4 that, if the flow rate is sufficiently high, [O2]el is negligibly small. For instance, with a flow rate of 1.00 mL/min, [O2]el can be calculated to be 6 × 10-7 M. In view of the lowest oxygen levels that can be obtained upon thorough deoxygenation with highly purified nitrogen gas, i.e., 6 × 10-6 M,12 such an extra contribution to the total oxygen concentration is clearly acceptable. In fact, an up to 10-fold higher current can be tolerated. Optimization of Switching Times. Evidently, only a minor fraction of the 200-µL sample volume, moving at a flow rate of 1388 Analytical Chemistry, Vol. 71, No. 7, April 1, 1999

1.00 mL/min can be injected into the CE capillary. Hence the time interval ∆(ts - ti) between switching of the six-way injection valve and starting the hydrodynamic injection had to be optimized with respect to both analyte response achievable in CE and precision. To determine the optimum value of the time interval, the fluorescence signal generated by a 7 × 10-5 M solution of 5/0/2 (which is not affected by the oxygen concentration in the buffer solution) was measured at different ∆(ts - ti) values, applying hydrodynamic injection either for 0.08 min at -30 mbar or for 0.16 min at -15 mbar. The optimum time intervals were 0.23 and 0.19 min, respectively. The precision was satisfactory with RSD values of 2.4% and 1.3% (n ) 10), respectively. All CE experiments were performed with the CE program of Table 1. Optimization of Detection Parameters. The optimum gating time for the luminescence detector, tg, was found to be 0.50 ms, and the optimum delay time, td, before the start of data sampling, 0.05 ms. A higher value of tg results in a reduction of the S/N ratio, because it only provides extra noise while no additional phosphorescence is sampled. A reduction of tg, self-evidently, results in a reduction of the measured phosphorescence intensity. With td < 0.05 ms, there is a higher background (reflected radiation and stray light from the source), which results in a lower signal-to-noise ratio, whereas higher td values cause a loss of phosphorescence signal. As far as sensitized phosphorescence is concerned, excitation via the short-wavelength absorption band at 230 nm is more appropriate (higher signal-to-noise ratio) than excitation via the long-wavelength band around 300 nm, despite the fact that the lamp output is higher and more stable at the latter wavelength. The reason is that at 300 nm the phosphorescence signal due to direct excitation of biacetyl contributes significantly to the background. Sensitized Phosphorescence Detection Mode. As is obvious from eq 1, the sensitized phosphorescence signal is proportional not only to the analyte concentration but also to the energytransfer efficiency, which will be dependent on the biacetyl concentration. Therefore, linear curves are expected with slopes that are dependent on the biacetyl concentration. This concentration also determines the noise level since the main cause of background comes from direct biacetyl phosphorescence. Measurements covering the concentration range 10-6-5 × 10-5 M for 0/0/1, 0/0/2,6, and 0/0/1,3,7, using moderate injection volumes (pt ) 144 mbar‚s), indeed revealed a linear dependence (R2 values of 0.999, 0.997, and 0.995, respectively, six data points). The detection limits (S/N ) 3) of the test analytes were calculated for biacetyl concentrations of 0.005, 0.01, 0.02, and 0.05 M (see Figure 5). To discuss the role of biacetyl more explicitly, the energytransfer efficiency introduced in eq 1 is written as8 -1 ϑDA ) kt[A]/(kt[A] + (τD t 0) )

(5)

wherein [A] represents the biacetyl concentration, kt is the rate constant for energy transfer from the analyte to biacetyl, and τD 0 is the analyte triplet lifetime in the absence of biacetyl. Equation 5 implies that the sensitized phosphorescence intensity will become higher when the concentration of biacetyl increases, until

Figure 5. Detection limits for 2,6-naphthalenedisulfonic acid (0/0/ 2,6) and 1,3,7-naphthalenetrisulfonic acid (0/0/1,3,7) (sensitized RTPL), and 4-amino-5-hydroxy-2,7-naphthalenedisulfonic acid (4/5/ 2,7) and 4,5-dihydroxy-2,7-naphthalenedisulfonic acid (0/4,5/2,7) (quenched RTPL) as a function of biacetyl concentration; p‚t ) 144 mbar‚s. For detection parameters, see text.

ϑDA t

approaches unity (see also eq 1). Unfortunately, the background noise is also enhanced with higher biacetyl concentrations. According to Figure 5, in the sensitized mode, a biacetyl concentration in the 0.005-0.02 M range is to be recommended. According to eqs 1 and 5, the sensitized phosphorescence intensity, Ip(sens), does not change significantly when 10% biacetyl is lost from the buffer solution, even if the triplet lifetime, τD 0 , is as short as 0.1 µs. Therefore, stable sensitized phosphorescence signals can be maintained during a whole working day. The LODs of 0/0/2,6 and 0/0/1,3,7 with 0.02 M biacetyl are 2 × 10-7 and 4 × 10-7 M, respectively. Under the same conditions, 0/0/1 has an LOD of ∼2 × 10-6 M. This relatively high LOD can only partly be explained by the lower extinction coefficient of 0/0/1 at 230 nm. One might speculate that for this compound the intersystem crossing efficiency, ϑD ISC, is less favorable; an is an alternative explanation. With higher extremely low τD 0 injection volumes (pt ) 768 mbar‚s), i.e., under stacking conditions, LODs ranging from 5 × 10-8 to 4 × 10-7 M were reached. Quenched Phosphorescence Detection Mode. As is obvious from eq 3, in quenched RTPL detection, linear graphs are only expected if I-1 is plotted as a function of analyte (quencher) concentration. Indeed, linear Stern-Volmer plots were obtained for 4/5/2,7 and 0/4,5/2,7 (R2 values of 0.999 and 0.998, respectively, six data points), using standards in the 2.5 × 10-6-5 × 10-5 M range and a moderate injection volume (pt ) 144 mbar‚ s). Because of the nonlinear response in this detection mode, the LODs for 4/5/2,7 and 0/4,5/2,7 were not determined by extrapolation but calculated from the S/N ratio of the standard with the lowest concentration. Figure 5 shows clearly that for the quenched phosphorescence detection mode the LODs tend to improve with an increasing biacetyl concentration. This can be explained by the lower relative noise of the direct biacetyl phosphorescence signal (I0 in eq 3) attained at a higher biacetyl concentration (shotnoise conditions). A 2-3-fold improvement of LODs is achieved in going from 0.02 to 0.05 M. Nevertheless, to analyze NScontaining samples, 0.02 M biacetyl is the concentration of choice; it is the optimum value if both sensitized and quenched phosphorescence detection are applied.

Equation 3 shows that the sensitivity of quenched RTPL is proportional to the bimolecular quenching rate constant, kq, as well as to τ0, the triplet lifetime in the absence of a quencher. If τ0 is known, kq can be determined from the slope (kqτ0) of the Stern-Volmer equation. The actual concentration [Q] of the quenching analytes during a CE run can be calculated from [Q]0s the concentration used for injectionsand the electrophoretic dilution factor, D. The latter equals the ratio of the length of the peak at the detection window and the length of the sample plug directly after hydrodynamic injection and is estimated to be 6 and 7 for 0/4,5/2,7 and 4/5/2,7, respectively. The triplet lifetime τ0 of biacetyl in the deoxygenated buffer system was measured to be 70 µs. From these data, the kq values of the substituted NS were calculated to be as high as ∼1010 M-1 s-1. This underlines that the quenching is diffusion-controlled, as is required for sensitive detection by quenched RTPL. Under stacking conditions (pt ) 768 mbar‚s), the LODs in the presence of 0.02 M biacetyl are ∼10-7 M. CONCLUSIONS Sensitized and quenched phosphorescenceswithout the use of micellesshas potential as a detection technique for CE. Detection is based on the room-temperature phosphorescence of biacetyl in liquid solutions. No pre- or postcapillary complexation reactions are required. A simple deoxygenation device can be used to achieve low oxygen levels in the buffer solution, and a homemade interface enables easy sample introduction. The instrumental setup can easily be extended to become a hyphenated LC-CE or solid-phase extraction (SPE)-CE system. This will be interesting because of the possibility of including on-line sample preparation or preconcentration procedures. Even though some parameters can be optimized further, the present detection limits of down to 5 × 10-8 M illustrate the inherent sensitivity of the technique. These can probably be improved further by modifying the optical system to enhance the photon flux in the CE capillary. For sensitized RTPL detection, a laser-based excitation scheme might enhance the performance, with regard to detection limits attainable. For quenched RTPL, it will be attempted to reduce the noise of I0 (see eq 3), i.e., of the baseline, to lower the LODs; a detection scheme that involves the quenching of NS-sensitized biacetyl phosphorescence will probably be appropriate for this purpose: under shot-noise conditions, the higher I0 achieved with this scheme will enhance the S/N ratio of the baseline signal. It should be noted that phosphorescence detection is more selective than fluorescence detection. If one deals with a class of analytes with T1-state energies close to that of biacetyl, as is the case with the NS studied in this work, the analytes within that class will provide either sensitized or quenched RTPL, which leads to additional selectivity. As far as applicability is concerned, sensitized RTPL can be used for all analytes that combine a nonzero triplet quantum yield with a sufficiently high T1-state energy, provided that steric hindrance plays no inhibiting role. It may be expected for instance that benzene derivatives meet these conditions. Preliminary experiments performed in our laboratory indeed show that, for example, benzoic acid, 2-chlorobenzoic acid, and 2,4-dichlorobenzoic acid can be detected by sensitized RTPL. Analytical Chemistry, Vol. 71, No. 7, April 1, 1999

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Dynamic phosphorescence quenching is not limited to analytes with low T1-state energies. As shown earlier by our group,21,22 a large variety of organic compounds and inorganic ions show high quenching rate constants as well, e.g. based on electron- and/or proton-transfer reactions. Also, for this RTPL mode, the selectivity and the applicability range have to be further explored. (21) Donkerbroek, J. J.; Veltkamp, A. C.; Gooijer, C.; Velthorst, N. H.; Frei, R. W. Anal. Chem. 1983, 55, 1886-1893. (22) Gooijer, C.; Markies, P. R.; Donkerbroek, J. J.; Velthorst, N. H.; Frei, R. W. J. Chromatogr. 1984, 289, 347-354.

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ACKNOWLEDGMENT The authors thank the Dutch Foundation for the Advancement of Science (NWO) for financial support.

Received for review October 15, 1998. Accepted January 5, 1999. AC981130H