Phosphorescence for Sensitive Enantioselective Detection in Chiral

Jul 8, 2009 - Phosphorescence for Sensitive Enantioselective Detection in Chiral Capillary Electrophoresis. Ivonne Lammers, Joost Buijs, Gert van der ...
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Anal. Chem. 2009, 81, 6226–6233

Phosphorescence for Sensitive Enantioselective Detection in Chiral Capillary Electrophoresis Ivonne Lammers, Joost Buijs, Gert van der Zwan, Freek Ariese,* and Cees Gooijer Department of Analytical Chemistry and Applied Spectroscopy, Laser Centre Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands Enantioselective phosphorescence lifetime detection was combined with chiral cyclodextrin-based electrokinetic chromatography for the analysis of camphorquinone (CQ). A time-gated detection system based on a pulsed lightemitting diode for excitation at 465 nm was developed for the online lifetime determination. The background electrolyte for the chiral separation consisted of 20 mM r-cyclodextrin (r-CD), 10 mM carboxymethyl-β-CD, and 25 mM borate buffer at pH 9.0. The separation of (+)CQ and (-)-CQ is caused by a difference in association constants of these enantiomers with r-CD. Under the separation conditions, different phosphorescence lifetimes were obtained for (+)-CQ and (-)-CQ (τ ) 384 ( 8 and 143 ( 5 µs, respectively), which could be used to distinguish the enantiomers. This selectivity in detection is based on a difference in protection of the enantiomers against phosphorescence quenching after their complexation with r-CD. Concentration detection limits were 2 × 10-7 and 1 × 10-6 M for (+)-CQ and (-)-CQ, respectively. After correction for the lifetime shortening by triplet-triplet annihilation at higher CQ concentrations, a linear dynamic range was obtained from the detection limit up to 2 mM. The system was used to determine the enantiomeric impurity levels of commercial samples of (+)-CQ and (-)-CQ; 0.2% and 0.1%, respectively. Currently, in biomedical and pharmaceutical analysis there is much interest in enantioselectivity since the two enantiomers of a chiral compound generally have different biological activities. For example, often only one enantiomer of a drug exhibits the desired pharmaceutical effect, whereas the other isomer may show unwanted side effects. For drugs that are to be administered enantiopure, the absence of enantiomeric impurities must be demonstrated down to the 0.1% level.1 Although enantioselective separation techniques (in liquid chromatography and capillary electrophoresis (CE)) are well-developed and very efficient, the available enantioselective detection methods are still far from ideal. This paper will focus on the latter problem. CE offers a number of advantages for the separation of enantiomers. The low sample and reagent consumption allows the use of expensive chiral selectors and enantiopure standards. * To whom correspondence should be addressed. Fax: 31 (0) 20 5987543. E-mail: [email protected]. (1) ICH Harmonized Tripartite Guidelines. Q3A(R2): Impurities in New Drug Substances. Fed. Regist. 2003, 68, 6924-6925.

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The wide choice of background electrolyte (BGE) compositions gives ample flexibility for optimization, including the use of different chiral selectors or combinations of these at various concentrations. Fast analyses with a high efficiency are possible. By far most enantioseparations are performed with cyclodextrins (CDs) as the chiral selector in cyclodextrin-based electrokinetic chromatography (CD-EKC).2-4 One of the drawbacks of CE is the difficulty of coupling this separation technique with chiroptical detection techniques. Enantioselective optical methods such as polarimetry and circular dichroism are based on a differential measurement and suffer from a limited sensitivity. This hampers their coupling to CE because of the short optical pathlengths (typically 50 or 75 µm) involved. Furthermore, most chiral separations in CE are still performed with a continuous BGE; the high concentration of chiral selector in the BGE that passes the detection window may cause a significant response with these enantioselective detection methods.5 Moreover, the response of enantiomers in these chiroptical detection techniques is equal in magnitude but opposite in sign. No detector response is observed for a pure racemate. Therefore, these chiroptical detection techniques cannot determine both the enantiomeric ratio and the absolute enantiomer concentrations at the same time in case of incomplete separations. Often they are combined with absorbance detection in order to obtain information about the total enantiomer concentration.6 Alternatively, the optical detection techniques fluorescence and phosphorescence, which measure analyte emission against a theoretically low background, may be considered for sensitive enantioselective detection in CE. In various chiral recognition studies, fluorescence techniques have been used for a wide range of enantiomers. Enantioselective responses were found in fluorescence intensity, spectral shifts, fluorescence polarization, fluorescence lifetimes, fluorescence resonance energy transfer, and fluorescence quenching.7-11 However, in many cases the (2) Chankvetadze, B. J. Chromatogr., A 2007, 1168, 45–70. (3) Juvancz, Z.; Kendrovics, R. B.; Iva´nyi, R.; Szente, L. Electrophoresis 2008, 29, 1701–1712. (4) Gu ¨ bitz, G.; Schmid, M. G. J. Chromatogr., A 2008, 1204, 140–156. (5) Chankvetadze, B. Electrophoresis 2002, 23, 4022–4035. (6) Bobbitt, D. R.; Linder, S. W. TrAC, Trends Anal. Chem. 2001, 20, 111– 123. (7) Pu, L. Chem. Rev. 2004, 104, 1687–1716. (8) Xu, Y.; McCarroll, M. E. J. Photochem. Photobiol., A 2007, 187, 139–145. (9) Williams, A. A.; Fakayode, S. O.; Lowry, M.; Warner, I. M. Chirality 2009, 21, 305–315. (10) Mei, X. F.; Martin, R. M.; Wolf, C. J. Org. Chem. 2006, 71, 2854–2861. (11) Marcelo, G.; de Francisco, R.; Gonza´lez-A´lvarez, M. J.; Mendicuti, F. J. Photochem. Photobiol., A 2008, 200, 114–125. 10.1021/ac900750e CCC: $40.75  2009 American Chemical Society Published on Web 07/08/2009

discrimination power was rather small. As far as we know, these enantioselective techniques have not yet been coupled to analytical separations. In this article we will focus on phosphorescence detection for the recognition of enantiomers. The potential enantioselectivity of liquid-state room temperature phosphorescence (RTP) in batch experiments has been demonstrated in the recent literature.12-15 This rather new enantioselective detection method is based on the complexation of analytes with CDs, which are homochiral. The interaction between a racemic guest and cyclodextrins may lead to the formation of diastereomeric associates with different photophysical properties. In favorable cases, the phosphorescence quantum yields and lifetimes are enantiomer-dependent. In contrast to polarimetry and circular dichroism, the distinct phosphorescence signals of the two enantiomers do not cancel each other. This enables the determination of the enantiomeric ratio and the absolute concentrations of the enantiomers at the same time. Two different approaches can be discerned for chiral discrimination in cyclodextrin-induced room-temperature phosphorescence (CD-RTP). For analytes exhibiting native phosphorescence, the complexation between the analyte and the CD can be sufficient as such.13 Sometimes a third component is needed as a heavy atom perturber and/or a space regulator.14,15 For nonphosphorescent analytes, a ternary complex between the analyte, CD, and a phosphorescent reagent can be used.12 In this work we will focus on the first approach: the enantioselective detection of phosphorescent analytes, using (+)-CQ and (-)camphorquinone (CQ) as model compounds. Batch time-resolved RTP experiments in our laboratory have shown that after complexation with R-CD, the two enantiomers of CQ have different phosphorescence intensities and lifetimes under deoxygenated conditions.13 It has been shown in the literature that R-CD and CQ form predominantly 2:1 complexes and that 1:1 complexes do not play an important role at the R-CD concentration considered. The equilibrium constant of the 2:1 complex is slightly larger for (+)-CQ than for (-)-CQ. The influence of the complexation with R-CD on the absorption spectrum of CQ is relatively small and equal for both enantiomers.16 Further, only small differences in fluorescence intensity were found between (+)-CQ and (-)-CQ in the presence of R-CD.13 This indicates that not the equilibrium constants as such but the degree of protection against phosphorescence quenching is responsible for the large enantiomeric difference in RTP. Apparently, in comparison to (-)-CQ, the complexation of (+)CQ with R-CD provides a better protection against the aqueous environment, which results in a longer phosphorescence lifetime and a higher intensity. In principle, the lifetime can be used for the discrimination between the enantiomers and the intensity for the quantification.13 The separation of CQ enantiomers by chiral CD-EKC, using a mixture of neutral R-CD and negatively charged carboxymethyl(12) García-Ruiz, C.; Hu, X.; Ariese, F.; Gooijer, C. Talanta 2005, 66, 634–640. (13) García-Ruiz, C.; Scholtes, M. J.; Ariese, F.; Gooijer, C. Talanta 2005, 66, 641–645. (14) Zhang, X. H.; Wang, Y.; Jin, W. J. Talanta 2007, 73, 938–942. (15) Zhang, X. H.; Wang, Y.; Jin, W. J. Anal. Chim. Acta 2008, 622, 157–162. (16) Bortolus, P.; Marconi, G.; Monti, S.; Mayer, B. J. Phys. Chem. A 2002, 106, 1686–1694.

Figure 1. Structures of the enantiomers of camphorquinone.

β-cyclodextrin (CM-β-CD), has been published.17 Detection was based on a quenched phosphorescence methodology in which a strongly phosphorescent compound, i.e., brominated naphthalene sulfonate, was added to the BGE. After deoxygenation, a continuous phosphorescence background was obtained. The CQ enantiomers induced dynamic quenching of this phosphorescence background, thereby causing negative peaks in the electropherogram. For both (+)-CQ and (-)-CQ a concentration detection limit of 7 × 10-7 M injected was achieved. No clear difference in peak heights was observed for (+)-CQ and (-)-CQ. Therefore, this quenched phosphorescence detection method should not be considered enantioselective. In this paper we report for the first time the coupling of a chiral CE separation to an enantioselective detection method based on CD-RTP for the analysis of (±)-CQ. The phosphorescence lifetime was used to distinguish the two enantiomers. A detection system was developed that enables the measurement of luminescence decays online during the recording of the electropherogram. Excitation was performed using a light-emitting diode (LED) at 465 nm, which has the advantages of small size, low costs, strong emission, near-monochromatic output, high stability, and longterm reliability.18 An important advantage of LEDs for their use in phosphorescence detection is the possibility to create blockshaped pulses at the microsecond level without substantial tailing. This enables the use of a short delay and creates “clean” phosphorescence decays. Following a detailed characterization of the setup for phosphorescence lifetime detection in CE, we will describe the influence of the BGE constituents on the steadystate and time-resolved phosphorescence and demonstrate the feasibility of this technique by determining the enantiomeric impurity levels in (+)-CQ and (-)-CQ standards. EXPERIMENTAL SECTION Chemicals. (±)-Camphorquinone, carboxymethyl-β-cyclodextrin sodium salt (degree of substitution ∼ 3), R-cyclodextrin, and sodium hydroxide were purchased from Fluka (Buchs, Switzerland). (1R)-(-)-Camphorquinone and (1S)-(+)-camphorquinone were obtained from Aldrich (Steinheim, Germany). Their structures are shown in Figure 1. Boric acid was obtained from Sigma (St. Louis, MO). All chemicals were used as received. Water was purified with a Milli-Q system from Millipore (Bedford, MA). Batch Experiments. The Cary Eclipse luminescence spectrometer (Varian, Melbourne, Australia) was used for batch experiments. The samples were purged with nitrogen in a capped, long-necked cuvette (Hellma, Mu¨llheim, Germany) via a stainless (17) García-Ruiz, C.; Siderius, M.; Ariese, F.; Gooijer, C. Anal. Chem. 2004, 76, 399–403. (18) Xiao, D.; Zhao, S.; Yuan, H.; Yang, X. Electrophoresis 2007, 28, 233–242.

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steel tube for deoxygenation of the samples.19 A stable nitrogen flow was maintained with a flow regulator. The temperature of the samples was kept at 20 °C using the single cell Peltier cooler of the spectrometer. All batch experiments were performed at an excitation wavelength of 460 nm with a pulsed Xe-lamp. The excitation and emission slit widths were set to 10 nm, and the PMT voltage was 1000 V. A 335-620 nm excitation filter, which was provided with the instrument, was inserted to prevent excitation of CQ by second order light. A 430-1100 nm filter was used as an emission filter. The phosphorescence emission spectra were recorded with a delay time of 100 µs and a gate time of 5000 µs. In contrast to the measured phosphorescence lifetimes, the intensities of the spectra depend on the delay settings. Therefore, all spectra were corrected for the signal loss during the delay time by multiplication with a correction factor depending on the phosphorescence lifetimes and (in the case of a biexponential curve) on the amplitudes belonging to the different lifetimes. For the lifetime measurements, excitation was performed at 460 nm and emission was detected at 563 nm. In total, 250 decays were averaged that were recorded with a gate time of 10 µs and increasing delay times from 50 to 5000 µs in 10 µs steps. Decay curves were fitted in OriginPro 8. Capillary Electrophoresis. Separations were achieved on a CE system (200 series) from Prince Technologies, (Emmen, The Netherlands) using an uncoated fused-silica capillary (Polymicro, Phoenix, AZ) with an inner diameter of 75 µm, an outer diameter of 375 µm, a total length of 100 cm, and an effective length of 60 cm. A modified buffer vial was used to purge the buffer solution continuously with nitrogen for the removal of oxygen.20 Also here the nitrogen flow rate was kept constant with a flow regulator. All running buffers and sample solutions were filtered with 0.2 µm syringe filters (Whatman, Dassel, Germany). Sample injections were performed by applying 100 mbar for 10 s. Separations were performed with 20 kV at room temperature. For all separations, a BGE of 25 mM borate buffer pH 9.0 with 20 mM R-CD and 10 mM CM-β-CD was used. All samples consisted of CQ dissolved in milli-Q water. At the beginning of a day, the capillary was rinsed for 15 min with 0.1 M NaOH (1 bar), 15 min with milli-Q water (1 bar) and 15 min with BGE (1 bar). Online Lifetime Measurements. A home-built detection system based on a backscattering configuration was used for the online recording of phosphorescence decay curves in CE. Figure 2 shows the setup schematically. A high-intensity light-emitting diode of 465 nm (ELJ-465-627, Roithner Lasertechnik, Vienna, Austria), connected to a home-built pulse generator with a constant current source typically set to 0.5 A, was used to obtain excitation pulses of 300 µs wide at a frequency of 230 Hz. The light emanating from the LED was passed through a 465 nm bandpass filter (FF01-465/30-25, Semrock, Rochester, NY), collimated by a lens and reflected under 90° by a 506 nm dichroic mirror (FF506Di02-25, Semrock, Rochester, NY). Subsequently, a quartz microscope objective (03-0304, Partec Optics, Mu¨nster, Germany) was used to focus the excitation light into the capillary center and to collect the phosphorescence. The emission was passed through the dichroic mirror, a 583 nm bandpass filter (FF01-583/ (19) Donkerbroek, J. J.; Elzas, J. J.; Gooijer, C.; Frei, R. W.; Velthorst, N. H. Talanta 1981, 28, 717–723. (20) Kuijt, J.; Roman, D. A.; Ariese, F.; Brinkman, U. A. T.; Gooijer, C. Anal. Chem. 2002, 74, 5139–5145.

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Figure 2. Scheme of the LED-induced phosphorescence detector for CE. See text for a detailed description.

120-25, Semrock, Rochester, NY), and a focusing lens. The light was subsequently detected with a photomultiplier tube (PMT) (H9656-03, Hamamatsu Photonics, Hamamatsu, Japan). For phosphorescence detection, a delay of 10 µs was set with respect to the negative edge of each LED pulse before starting to record the luminescence decays. A home-built pulse burst generator was used to trigger the A/D converter (PCI-1742U, Advantech, Vienna, Austria) for the acquisition of the signal from the PMT each 2 µs during a total gate of 1024 µs. The decays were written to the computer and averaged each second. The intensity was obtained by averaging the signals acquired during the entire gate. In order to minimize CE-induced heating effects at the detection site, the capillary was cooled with a Peltier element set to -10 °C (CP1.071-06, Melcor, Trenton, NJ) 2 cm before and after the detection window. This Peltier was driven by a home-built temperature controller. The decay curves were fitted with a monoexponential decay in Gnuplot 4.2.4 (open source software). For fluorescence detection, the pulse width was set to 1 ms, the LED current to 0.35 A, the triggering was performed on the positive edge of the light pulse without a delay, and the gate width was 1024 µs. No fluorescence decays were recorded. RESULTS AND DISCUSSION The use of CDs as chiral selectors in CD-EKC makes this separation technique in principle suited for coupling to enantioselective CD-RTP detection. Batch experiments showed large lifetime differences between the phosphorescence of (+)-CQ and (-)-CQ in aqueous solutions containing 20 mM R-CD.13 However, for the electrophoretic separation of the two uncharged CQ enantiomers, a dual cyclodextrin system was needed. Negatively charged carboxymethyl-β-cyclodextrin (CM-β-CD) was added as the carrier of CQ, while the neutral R-CD acted as the chiral selector. Experiments with only R-CD resulted in the migration of both enantiomers with the electroosmotic flow (EOF), whereas CM-β-CD alone did not separate the two enantiomers from each other.17 Batch Experiments. In order to investigate the influence of the different BGE constituents on the CQ phosphorescence, batch experiments were performed under deoxygenated conditions.

Figure 3. Phosphorescence spectra of 0.2 mM (+)-CQ (black solid line), 0.2 mM (()-CQ (0.1 mM (+)-CQ and 0.1 mM (-)-CQ; red dashed line), and 0.2 mM (-)-CQ (blue dotted line) in 25 mM borate buffer pH 9.0 and (a) 20 mM R-CD, (b) 10 mM CM-β-CD, and (c) 20 mM R-CD and 10 mM CM-β-CD. All samples were purged with nitrogen to remove oxygen. The phosphorescence intensities on the vertical axis are given in arbitrary units. Table 1. Phosphorescence Lifetimes (Microseconds) and Corresponding Amplitudes (Arbitrary Units) Obtained from the Batch Samples Shown in Figure 3 20 mM R-CD A1

τ1

A2

τ2

(+)-CQ 26 437 (±)-CQ 14 414 13 116 (-)-CQ 25 120

10 mM CM-β-CD

20 mM R-CD and 10 mM CM-β-CD

A1

τ1

A1

τ1

A2

τ2

20 20 20

106 111 114

18 10 23

385 356 120

7 14

134 109

Figure 3 shows the phosphorescence emission spectra corrected for the delay time of enantiopure or racemic CQ in the presence of 20 mM R-CD, 10 mM CM-β-CD, or both CDs. Table 1 lists the corresponding lifetimes and amplitudes. All samples were buffered with 25 mM borate at pH 9.0. This buffer did not influence the amplitudes and lifetimes obtained (data not shown). Excitation was performed at 460 nm, close to the maximum of the S0-S1 absorption band of CQ. Hardly any phosphorescence was observed in samples that did not contain CDs (data not shown). The enantioselectivity of CD-RTP is obvious from Figure 3a. After complexation with R-CD and deoxygenation, a high-intensity phosphorescence signal of CQ is observed with strongly different signal heights and lifetimes for the two enantiomers in accordance with earlier literature.13 The amplitude of (+)-CQ is slightly higher than the amplitude of (-)-CQ for both the enantiopure and the racemic samples (see Table 1). This indicates that the concentration of 〈R-CD|CQ|R-CD〉 is a little higher for (+)-CQ than for (-)CQ, which is in qualitative agreement with its higher equilibrium constant reported by Bortolus et al.16 However, the enantioselectivity of the phosphorescence signal can be almost completely attributed to the phosphorescence lifetimes, which differ by a factor of 3.6. Apparently, the (+)-enantiomer is much better protected against solvent-induced quenching than (-)-CQ. The presence of a phosphorescence signal in the sample containing only CM-β-CD (shown in Figure 3b) indicates the complexation of both (+)-CQ and (-)-CQ with this modified CD. The phosphorescence lifetimes found in the presence of CM-βCD are shorter than those in the presence of R-CD, especially for (+)-CQ. Apparently, the 1:1 complexation of (+)-CQ with CM-βCD protects the phosphorophore less well against quenching by the aqueous environment than its 2:1 complex with R-CD.16 In contrast to the samples with R-CD, only a minor difference in lifetime is found for the two enantiomers: for (-)-CQ it is slightly

higher than for (+)-CQ. Clearly, this difference in lifetime is too small for enantiomeric discrimination, which is reflected in the fact that for the racemic sample only a single lifetime could be found. To the best of our knowledge the complexation constants of (+)-CQ and (-)-CQ with this modified β-CD have not yet been reported in the literature, but they have been published for the unmodified β-CD.16 The amplitudes in Table 1 indicate that the equilibrium constants for the formation of 1:1 inclusion complexes with CM-β-CD do not differ significantly for the two enantiomers, which agrees with the observation that (+)-CQ and (-)-CQ cannot be separated in CE when using exclusively CM-β-CD.17 Comparison of the phosphorescence intensities of (+)-CQ and (-)-CQ in the presence of 10 mM β-CD or CM-β-CD showed a 12% lower degree of complexation with the modified CD for both enantiomers (data not shown). On the basis of this difference and the literature values for β-CD, an equilibrium constant of 190 ± 50 M-1 could be calculated for the 1:1 complex formation between (±)-CQ and CM-β-CD. This was further supported by fitting the migration time shift of (±)-CQ in CE as a function of the CM-β-CD concentration in the BGE (data not shown). The batch phosphorescence spectra obtained under the solution conditions applied in CE (with both R-CD and CM-βCD) are depicted in Figure 3c. In these samples CQ can form complexes with both CDs. Clearly, the phosphorescence intensity and lifetime still offer a high degree of enantioselectivity, though somewhat lower than in the presence of only R-CD (Figure 3a). The lifetimes of (-)-CQ in solutions containing only R-CD or only CM-β-CD are very similar and therefore one “average” lifetime is found for (-)-CQ in the presence of both CDs. For (+)-CQ two different lifetimes were found, a long lifetime of 385 µs from its complexation with R-CD and a short lifetime of 134 µs from its complexation with CMβ-CD. Compared to the lifetimes of the samples of (+)-CQ with only R-CD or only CM-β-CD, the long lifetime decreased and the short lifetime increased somewhat, which is as yet not fully understood. Perhaps this is due to an energy transfer from the longer living 〈R-CD|(+)-CQ|R-CD〉 complex to the shorter living 〈CM-β-CD|(+)-CQ〉 complex. The amplitudes observed for (+)CQ show a ratio of 18:7 for the 〈R-CD|CQ|R-CD〉 versus the 〈CM-β-CD|CQ〉 complex, which agrees with the literature value for the (+)-CQ complexation with R-CD (K〈R-CD|(+)-CQ|R-CD〉 ) 14 100 ± 1700 M-2)16 and the 190 ± 50 M-1 value for its Analytical Chemistry, Vol. 81, No. 15, August 1, 2009

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Figure 4. (a) Electropherograms of the separation of 0.2 mM (()-CQ using a BGE of 25 mM borate buffer pH 9.0, 20 mM R-CD, and 10 mM CM-β-CD. The detection was performed with fluorescence (upper red trace and right intensity scale) and phosphorescence (lower black trace and left scale). (b and d) Online phosphorescence decay traces of (+)-CQ and (-)-CQ, respectively. (c and e) Residuals of a monoexponential decay fit for (+)-CQ and (-)-CQ, respectively. The BGE was purged with nitrogen to remove oxygen. The phosphorescence, fluorescence and residuals on the vertical axis are given in arbitrary units; delays in microseconds.

complexation with CM-β-CD as reported above. The long lifetime component of 356 µs found in the racemic sample relates to the complexation of (+)-CQ with R-CD. The nominal concentration of (+)-CQ in this mixture is 2 times less than in the enantiopure sample. This is reasonably well reflected in the amplitude of 10 belonging to the long lifetime in the racemic mixture, which is almost half the amplitude of 18 in the enantiopure sample (see Table 1). Furthermore, the amplitude of 14 for the short lifetime of 109 µs in the racemic mixture, which contains the contributions of the complex of (+)-CQ with CM-β-CD and the complexes of (-)CQ with CM-β-CD or R-CD, corresponds well with the sum of the corresponding halved amplitudes (3.5 + 11.5). We found no clear evidence for the formation of a mixed complex of CQ with both R-CD and CM-β-CD. The data obtained agree well with the assumption that only complexes with either R-CD or CM-β-CD are formed. Note that all emission spectra in Figure 3a-c show, in addition to the RTP, a weak band around 500 nm, which is the wavelength maximum of CQ fluorescence. However, this band is not caused by conventional fluorescence because the spectra were obtained using gated detection with a delay of 100 µs, long enough to avoid the detection of fluorescence or background scattering. Moreover, the band observed around 500 nm was found to have a lifetime identical to the phosphorescence lifetime, an intensity linearly proportional to the excitation power and to the CQ concentration, and a relative intensity with respect to the phosphorescence that increases with temperature. It is ascribed to thermally activated delayed fluorescence21 (a topic beyond the discussion in the present paper). Note that the concentrations of inclusion complexes with R-CD or CM-β-CD are very similar for both (+)-CQ and (-)-CQ because the complexation reaction is almost driven to completeness at the high CD concentrations used. This is reflected in the similar amplitudes found for both enantiomers in Table 1. However, the difference between the phosphorescence lifetimes of the two enantiomers is independent of the inclusion complex concentra(21) Barltrop, J. A.; Coyle, J. D. Principles of Photochemistry; John Wiley & Sons: Chichester, U.K., 1978.

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tions and is therefore not determined by the equilibrium constants. The lifetime difference is caused by a differential protection against phosphorescence quenching that the enantiomers experience when complexed with R-CD. In the literature several reasons can be found for such a difference in protection. The complexes of the enantiomers with cyclodextrins can have different complex geometries, which, for example, has been studied by NMR for the complexation of camphor with R-CD.22 Possible differences in conformational flexibility and structural motion may also play a role.23,24 Finally, a difference in dissociation rate of the inclusion complexes with (+)-CQ and (-)-CQ can cause a difference in phosphorescence yield if this rate is of the same order as the phosphorescence lifetime.13,16 On the basis of the current experimental data, we cannot distinguish between these processes. Capillary Electrophoresis. Following the characterization of the various complexes in batch, the enantioselective phosphorescence detection method was coupled online to CE using the detection system illustrated in Figure 2. For the CE analysis of (+)-CQ and (-)-CQ, a dual cyclodextrin system with both R-CD and CM-β-CD is used. The separation of the enantiomers is based on the difference between the equilibrium constants for the inclusion of (+)-CQ and (-)-CQ in R-CD.16 The enantiomers can be detected by phosphorescence (after deoxygenation of the BGE) or fluorescence, either with time-gated detection or by measuring during the pulse, respectively. For comparison, both electropherograms obtained upon injection of a racemic mixture of CQ are shown in Figure 4a. In the case of fluorescence detection there is a strong background, mainly caused by the scatter of excitation light from the capillary. This background is strongly reduced in RTP because of the applied delay in detection. In the fluorescence mode, two signals of almost equal area are obtained. The minor difference in intensity between (+)-CQ and (-)-CQ arises from the small difference in their degree of complexation, while both enantiomers have the same fluorescence quantum yields.13 The two signals obtained by phosphorescence detection, however, (22) Dodziuk, H.; Ejchart, A.; Lukin, O.; Vysotsky, M. O. J. Org. Chem. 1999, 64, 1503–1507. (23) Grabner, G.; Rechthaler, K.; Mayer, B.; Ko ¨hler, G.; Rotkiewicz, K. J. Phys. Chem. A 2000, 104, 1365–1376. (24) Connors, K. A. Chem. Rev. 1997, 97, 1325–1357.

Figure 6. (a) Phosphorescence lifetimes (microseconds), (b) peak areas in arbitrary units, and (c) peak areas corrected for the lifetime at the peak top as obtained from electrophoretic analysis as a function of injected CQ concentration (millimolar). The BGE was purged with nitrogen to remove oxygen. Figure 5. Phosphorescence intensities (black line) and lifetimes (red squares) during the migration of (+)-CQ and (-)-CQ for different analyte concentrations and LED powers: (a) 2 mM (()-CQ and 0.5 A LED current, (b) 0.2 mM (()-CQ and 0.5 A LED current, and (c) 2 mM (()-CQ and 0.05 A LED current. The lifetimes are only shown during the elution of the peaks; no meaningful lifetimes could be fitted in the absence of analyte. The BGE was purged with nitrogen to remove oxygen. The phosphorescence intensities on the vertical axis are given in arbitrary units; lifetimes in microseconds.

differ strongly in intensity, indicating an enantioselective response. The intensity of (+)-CQ, which migrates first, is a factor of 3.9 higher than that of (-)-CQ. Considering the fact that the concentrations of (+)-CQ and (-)-CQ in the racemic sample are the same and that the difference in their degrees of complexation with R-CD or CM-β-CD is only minor (see fluorescence trace of Figure 4a and the amplitudes in Table 1), this difference in RTP intensity must be caused predominantly by a difference in phosphorescence quantum yield and lifetime. Indeed, monoexponential analysis of the luminescence decays that were recorded online during the analysis of 0.2 mM (±)-CQ showed a lifetime of 400 µs for (+)-CQ and a lifetime of 149 µs for (-)-CQ. In parts b and d of Figure 4, the luminescence decays obtained for (+)-CQ and (-)-CQ at the electrophoretic maximum are depicted. In contrast to the batch experiments, the curves of (+)-CQ were fitted with a monoexponential decay. This was necessary due to the higher noise level in the online CE lifetime measurements caused by the shorter measurement times during the separation. It should be realized that during the electrophoretic migration of an analyte its concentration changes. Therefore, the concentration dependence of the phosphorescence lifetimes was investigated by analyzing the decay curves that are obtained each second during migration. As shown in Figure 5a, the lifetime of (+)-CQ decreases at higher concentrations: a clear dip in the lifetime is

observed at the center of the electrophoretic peak. This effect is absent at lower (+)-CQ concentration and also at lower LED power (parts b and c of Figure 5). These observations indicate that the effect requires high concentrations of the (+)-CQ complex with R-CD in the T1-state. Therefore, most probably it can be attributed to triplet-triplet annihilation.25 In agreement with this interpretation, the lifetime of (-)-CQ is less affected by this phenomenon (see Figure 5a). The probability that two (-)CQ molecules in the triplet state annihilate each other is smaller because of their shorter lifetimes. For the analytical discrimination between the enantiomers, the characteristic lifetime should of course be independent of the sample concentration. This can be established by determining the maximum phosphorescence lifetime during the migration of (+)CQ. This maximum is found at the flanks of the peak, where the analyte concentration is still low and triplet-triplet annihilation does not play a role. Figure 6a shows the online lifetime of (-)CQ at the peak maximum and the lifetime of (+)-CQ determined at the peak maximum and at the flanks as a function of injected analyte concentration. Because of triplet-triplet annihilation, the lifetime of (+)-CQ determined at the peak maximum decreases with increasing concentrations. As a result there is only a very small difference in lifetime left between (+)-CQ and (-)-CQ at 2 mM. However, the maximum lifetime of (+)-CQ, found at the flank of the migration peak, is constant around 384 ± 8 µs (n ) 12) and provides a good means for its discrimination from (-)-CQ, which has a lifetime of 143 ± 5 µs (n ) 12). (25) Kuijt, J.; Ariese, F.; Brinkman, U. A. T.; Gooijer, C. Electrophoresis 2003, 24, 1193–1199.

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The reduction of the phosphorescence lifetime of (+)-CQ in Figure 6a from τ0 (the subscript 0 indicates the absence of triplet-triplet annihilation) to τ with higher analyte concentrations can be described with the Stern-Volmer equation:21

τ)

1 τ0-1 + kan[T1]

(1)

where kan is the bimolecular rate constant of the annihilation reaction and [T1] the concentration of CQ in the T1 state. In a first approximation [T1] is proportional to the CQ-concentration and to the LED-power applied. Of course a full description of the curve would require a more detailed treatment of triplet-triplet annihilation, accounting for instance for the time dependency of the T1-state concentration. The area of the (+)-CQ and (-)-CQ electrophoretic peaks as a function of concentration is shown in Figure 6b. As expected, these plots deviate from linearity as a result of triplet-triplet annihilation, especially for (+)-CQ. Since the phosphorescence signal is proportional to τ, division of the intensity by the observed lifetime should result in a linear plot. This lifetime, however, varies continuously during the electrophoretic migration of the analyte. Nevertheless, and quite important from an analytical point of view, a straightforward correction of the peak areas by correction with the lifetime at the peak maximum gives linear calibration curves, as is shown in Figure 6c. No significant difference in slope was found for calibration lines obtained from enantiopure or racemic camphorquinone after its separation, indicating a good apparent recovery.26 In principle, a deviation from linearity of the calibration curves might be caused by the change in [CQ] versus [CD] ratio since the phosphorescence signals result from the inclusion complexes of CQ with the CDs. A linear calibration curve will only be obtained if the CDs are present in large excess. On the basis of the equilibrium constants given by Bortolus et al.,16 this deviation would only amount to a few percent at these concentrations. In comparison to triplet-triplet annihilation, its contribution can be neglected. The concentration limits of detection (LOD) for (+)-CQ and (-)-CQ are 2 × 10-7 and 1 × 10-6 M injected, respectively. The latter is less favorable because of the shorter phosphorescence lifetime of complexed (-)-CQ. In view of the low extinction coefficient of CQ at 465 nm (determined as 36 M-1 cm-1) and the small detection volume in the capillary, these LODs can be considered as very good. Distinction between the two enantiomers based on their phosphorescence lifetimes requires higher sample concentrations. With the online setup, the characteristic lifetimes of (+)-CQ and (-)-CQ in the presence of R-CD and CM-β-CD can be determined at concentrations higher than 1 × 10-5 M, which is therefore called the “limit of discrimination”. At lower concentrations, the decay curves are too noisy for precise lifetime determination. Furthermore, the decay of the very weak remaining background luminescence starts to interfere at low analyte levels. This background is probably caused by luminescence from the bandpass filters used. (26) Burns, D. T.; Danzer, K.; Townshend, A. Pure Appl. Chem. 2002, 74, 2201– 2205.

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The cyclodextrin concentrations used were based on optimized conditions for the electrophoretic separation of CQ.17 These excess concentrations also drive the complexation toward completeness, which makes the detection method more robust. A factor that might cause variations in the observed phosphorescence lifetimes is the electrophoretic current during a CE separation. For example an increase in buffer concentration, a higher separation voltage, a larger capillary i.d., and a decrease in capillary length all lead to a higher current and thus to more oxygen production by electrolysis of water in the inlet vial. This oxygen will affect the phosphorescence lifetimes and signal intensities through increased quenching. Also the temperature at the detection window (depending on the Joule heating, cooling by the Peltier element, and the LED power) will affect the phosphorescence lifetimes. Furthermore, a larger variation of the concentration of R-CD and/or CM-β-CD in the BGE will not only influence the separation obtained but also the phosphorescence signals and lifetimes. Therefore, care should be taken to keep the experimental conditions as constant as possible. Of course, the methodology for enantioselective phosphorescence detection in its current form can only be applied to phosphorescent analytes. For nonphosphorescent analytes, a labeling procedure or a ternary complex formation between the analyte, a phosphorophore, and a cyclodextrin12 might be anticipated. The equilibrium between the analyte and the cyclodextrins should be reached fast enough as in the case of camphorquinone to ensure that the observed signal intensities and lifetimes are independent of the migration time (data not shown). Enantioselective phosphorescence studies based on the formation of aggregates may therefore be more difficult to couple with a CE separation.27 Furthermore, it is not yet possible to predict the lifetime of a (chiral) phosphorescent molecule complexed with a cyclodextrin, for example, by modeling. Therefore, the pure enantiomers are still needed as reference material to determine the characteristic phosphorescence lifetime for a particular analyte. Parts a and b of Figure 7 show the determination of the enantiomeric impurity of commercially available (-)- and (+)CQ standards using the standard addition method. After electrophoretic analysis of 2 mM (-)-CQ, a clear signal is seen in Figure 7a for (+)-CQ, indicating the presence of an enantiomeric impurity in this standard. The time-resolved nature of this detection technique can be used to improve the electrophoretic resolution. Increasing the gate delay causes a relatively smaller signal decrease for the long-lived (+)-CQ than for (-)-CQ, which improves the selectivity for this impurity with respect to the overwhelming (-)-CQ signal. After a delay of 900 µs, only the (+)-CQ signal is visible, while the signal of (-)-CQ is almost absent (see insert Figure 7a). The reverse situation, i.e., the analysis of small concentrations of (-)-CQ in the (+)-CQ standard, is expected to be more difficult, because (-)-CQ has a longer migration time and more importantly a lower RTP response. Nonetheless, Figure 7b demonstrates that also the enantiomeric impurity in the (+)-CQ standard could still be easily detected. In order to estimate the concentration of the impurity, the peak areas were compared to samples of 2 mM (-)-CQ and (+)-CQ with an added enantiomeric impurity of 1%. The calibration curve is still linear at these impurity levels, because triplet-triplet annihilation (27) He, Y.; Fu, P.; Shen, X.; Gao, H. Micron 2008, 39, 495–516.

Figure 7. (a) Electropherogram of 2 mM (-)-CQ before (red dashed line) and after (black solid line) adding an enantiomeric impurity of (+)-CQ of 1%; the insert shows the same separation obtained with a detector delay of 900 µs. (b) Electropherogram of 2 mM (+)-CQ before (red dashed line) and after (black solid line) adding an enantiomeric impurity of (-)-CQ of 1%. The BGE was purged with nitrogen to remove oxygen. The phosphorescence intensities on the vertical axis are given in arbitrary units.

does not play a role at these low concentrations. For the standard of (-)-CQ, the enantiomeric impurity was approximately 0.1% whereas the enantiomeric impurity in (+)-CQ was estimated to be 0.2%. At the low impurity levels, lifetimes could not be determined for the minor enantiomer. CONCLUSIONS AND PERSPECTIVES The results outlined above demonstrate that the CDs present in the BGE to accomplish chiral separations in CE can also be useful for achieving enantioselectivity in phosphorescence detection. Direct CD-RTP detection of CQ can be used for the quanti-

fication and discrimination of its enantiomers after their separation. Note that the difference in complexation constants of the two enantiomers with R-CD causes their separation in CE but not the enantioselectivity in the optical detection. The difference in lifetimes can be attributed to the difference in protection against phosphorescence quenching of the CQ enantiomers by R-CD. For long phosphorescence lifetimes, at high analyte concentrations and under intense irradiation conditions, the triplet state concentrations are no longer negligible and triplet-triplet annihilation should be accounted for. Correction for this process results in a linear calibration curve based on the electrophoretic peak areas. The lifetime maxima at the flanks of the migration peaks of (+)CQ and (-)-CQ are characteristic for the enantiomers and can be used for discrimination purposes. This strategy will be very useful in the case of poorly reproducible migration times in CE or in the case of poorly separated enantiomers. In comparison with batch experiments, much lower levels of a minor enantiomer can be detected by the online CE-phosphorescence system. Ways to further improve the detection limits by indirect excitation of camphorquinone are currently being evaluated. In addition, other chiral analytes will be the subject of future investigations. ACKNOWLEDGMENT The authors thank Dick van Iperen and Roald Boegschoten for designing and constructing the Peltier cooling element and the Dutch Foundation for the Advancement of Science (NWOCW) for financial support (ECHO Grant No. 700.55.014).

Received for review April 7, 2009. Accepted June 22, 2009. AC900750E

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