Anal. Chem. 2010, 82, 9410–9417
Sensitized Enantioselective Laser-Induced Phosphorescence Detection in Chiral Capillary Electrophoresis Ivonne Lammers, Joost Buijs, Freek Ariese,* and Cees Gooijer Department of Biomolecular Analysis and Spectroscopy, Laser Centre, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands The sensitivity of enantioselective cyclodextrin-induced room-temperature phosphorescence detection of camphorquinone (CQ) is enhanced using sensitization via a donor with a high extinction coefficient. The enantiomeric distinction is based on the different phosphorescence lifetimes of (+)-CQ and (-)-CQ after their complexation with r-cyclodextrin (r-CD). The collisional Dexter energy transfer from the selected donor 2,6-naphthalenedisulfonic acid (2,6-NS) to the acceptor CQ is still very efficient despite the inclusion of the acceptor into CD. For coupling to the chiral separation of (()-CQ in cyclodextrin-based electrokinetic chromatography, the donor was added to the deoxygenated background electrolyte that consisted of 20 mM r-CD, 10 mM carboxymethyl-β-CD, and 25 mM borate buffer at pH 9.0. Time-resolved batch studies on sensitized phosphorescence show a significant enantioselectivity for (+)- and (-)-CQ in the presence of both r-CD and CM-β-CD although the lifetime difference is somewhat reduced with respect to direct excitation. The enantiomers were distinguished after their separation using an online time-resolved detection system. Excitation was performed at 266 nm with a pulsed, small-sized, quadrupled Nd: YAG laser. With 1 × 10-5 M 2,6-NS, limits of detection of 4.1 × 10-8 M and 5.2 × 10-8 M were found for (+)CQ and (-)-CQ, respectively. The online measured lifetimes were 238 ( 8 µs for (+)-CQ and 126 ( 10 µs for (-)-CQ. The method was used to determine the concentration of (()-CQ leaching from a cured dental resin into water. The extracts contained 4.7 ( 0.1 × 10-7 M of both (+)-CQ and (-)-CQ. Enantiomers may show completely different behaviors in living organisms such as differences in uptake, distribution, metabolism, excretion, toxicity, and pharmaceutical effects.1 Chirality is, therefore, very important in many fields of analytical chemistry including biomedical, pharmaceutical, environmental, agricultural, and food analysis.2,3 For example, in dentistry, the chiral compound camphorquinone (CQ) is often used as a photoinitiator for * To whom correspondence should be addressed. Fax: +31 (0) 20 5987543. E-mail:
[email protected]. (1) Smith, S. W. Toxicol. Sci. 2009, 110, 4–30. (2) Kumar, A. P.; Jin, D.; Lee, Y. I. Appl. Spectrosc. Rev. 2009, 44, 267–316. (3) Ward, T. J.; Baker, B. A. Anal. Chem. 2008, 80, 4363–4372.
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the polymerization of visible light-cured restorative resins.4 The leaching of CQ from cured or uncured composite resins has recently been investigated with gas or liquid chromatography coupled to mass spectrometry.5-7 However, no distinction was made between the two enantiomers of CQ although these might have different effects in living systems. For example, the degradation of (-)-CQ is significantly slower than the degradation of its mirror image (+)-CQ in yeast and rabbits.8,9 Here, we demonstrate the enantioselective analysis of (±)-CQ extracted from a cured composite resin using capillary electrophoresis (CE) and sensitized enantioselective phosphorescence detection. This novel analytical approach increases the detection sensitivity considerably, while the enantioselectivity is retained. Cyclodextrin-induced room-temperature phosphorescence (CDRTP) can be used as an enantioselective detection technique.10-12 For example, the formation of 2:1 (host/guest) inclusion complexes of R-cyclodextrin (R-CD) with (+)-CQ or (-)-CQ provides a different degree of protection against quenching in deoxygenated aqueous samples. This results in a substantial difference between (+)-CQ and (-)-CQ regarding their phosphorescence intensity and lifetime.11 Recently, we showed the coupling of this detection method to cyclodextrin-based electrokinetic chromatography (CDEKC) to distinguish the CQ enantiomers based on both migration time and phosphorescence lifetime.13 For the chiral separation of (±)-CQ, a dual cyclodextrin system is needed consisting of neutral R-CD and negatively charged carboxymethyl-β-cyclodextrin (CM-β-CD).8 In contrast to R-CD, no enantioselective RTP signal is obtained for the diastereomeric complexes of (+)-CQ or (-)-CQ with CM-β-CD. However, the difference between the phosphorescence decay curves of the two enantiomers in a (4) Van Landuyt, K. L.; Snauwaert, J.; de Munck, J.; Peumans, M.; Yoshida, Y.; Poitevin, A.; Coutinho, E.; Suzuki, K.; Lambrechts, P.; van Meerbeek, B. Biomaterials 2007, 28, 3757–3785. (5) Alvim, H. H.; Alecio, A. C.; Vasconcellos, W. A.; Furlan, M.; de Oliveira, J. E.; Saad, J. R. C. Dent. Mater. 2007, 23, 1245–1249. (6) Michelsen, V. B.; Moe, G.; Skålevikc, R.; Jensen, E.; Lygre, H. J. Chromatogr., B 2007, 850, 83–91. (7) Rogalewicz, R.; Batko, K.; Voelkel, A. J. Environ. Monit. 2006, 8, 750– 758. (8) García-Ruiz, C.; Siderius, M.; Ariese, F.; Gooijer, C. Anal. Chem. 2004, 76, 399–403. (9) Robertson, J. S.; Hussain, M. Biochem. J. 1969, 113, 57–65. (10) García-Ruiz, C.; Hu, X.; Ariese, F.; Gooijer, C. Talanta 2005, 66, 634–640. (11) García-Ruiz, C.; Scholtes, M. J.; Ariese, F.; Gooijer, C. Talanta 2005, 66, 641–645. (12) Zhang, X. H.; Wang, Y.; Jin, W. J. Anal. Chim. Acta 2008, 622, 157–162. (13) Lammers, I.; Buijs, J.; van der Zwan, G.; Ariese, F.; Gooijer, C. Anal. Chem. 2009, 81, 6226–6233. 10.1021/ac101764z 2010 American Chemical Society Published on Web 10/21/2010
mixture of R-CD and CM-β-CD after direct excitation is still large enough to distinguish between them during their electrophoretic migration.13 Previously, direct excitation with a light-emitting diode (LED) at 465 nm was used to obtain enantiomer-dependent lifetimes during the CE separation of (±)-CQ. Despite the low extinction coefficient of CQ at this wavelength, the limits of detection (LOD) of 2 × 10-7 M for (+)-CQ and 1 × 10-6 M for (-)-CQ were still reasonable due to the high excitation power of the LED and the efficient light collection.13 Nevertheless, indirect excitation of CQ using energy transfer from a donor possessing more favorable absorption characteristics may improve the sensitivity even further. For example, sensitized RTP coupled to separation techniques has been shown to enhance the phosphorescence emission of biacetyl, which has an R-diketone chromophoric group with low extinction coefficients similar to CQ.14-19 Resonance energy transfer can be based on two different mechanisms. Fo¨rster described the nonradiative transfer of excited-state energy based on dipole-dipole interactions, which can occur over relatively long distances (∼8-10 nm). This Coulombic mechanism is only predominant for allowed transitions. Sensitized phosphorescence, however, is based on triplet-triplet energy transfer indicated as D*(T1) + A(S0) f D(S0) + A*(T1) in which D is the donor, A is the acceptor, S0 is the singlet electronic ground state, and T1 is the lowest triplet electronic excited state. The transitions of the donor and acceptor are both spin-forbidden and Fo ¨rster energy transfer will not occur. Under these conditions, energy transfer will be based on the exchange of electrons between the donor and acceptor. This Dexter energy transfer is only operative at short distances (
