Anal. Chem. 2002, 74, 3005-3012
Use of a Modified Cyclodextrin Host for the Enantioselective Detection of a Halogenated Diether as Chiral Guest via Optical and Electrical Transducers Birgit Kieser,† Christopher Fietzek,† Roswitha Schmidt,‡ Georg Belge,† Udo Weimar,† Volker Schurig,*,‡ and Gu 1 nter Gauglitz*,†
Institute of Physical and Theoretical Chemistry, University of Tu¨bingen, Auf der Morgenstelle 8, 72076 Tu¨bingen, Germany, and Institute of Organic Chemistry, University of Tu¨bingen, Auf der Morgenstelle 18, 72076 Tu¨bingen, Germany
In an alkaline rebreathing circuit, the inhalation anesthetic sevoflurane degrades into at least two products, one of them being the chiral halodiether 1,1,1,3,3-pentafluoro2-(fluoromethoxy)-3-methoxypropane (halodiether B). Using octakis(3-O-butanoyl-2,6-di-O-n-pentyl)-γ-cyclodextrin (Lipodex E) as chiral host diluted in the polysiloxane PS255, an exceptional large chiral separation factor r of 9.7 at 30 °C was found for halodiether B by capillary gas chromatography (cGC). Hence, the interaction of the single enantiomers and the racemic mixture of the halodiether B with Lipodex E was selected as a model system to study the enantioselective recognition by thickness shear mode resonators (TSMR), surface acoustic wave sensors, surface plasmon resonance (SPR), and reflectometric interference spectroscopy. Further investigations of the recognition process by using chemical sensors confirmed the preferential enrichment of the S-enantiomer resulting in 9-fold higher signals. Based on the distinction between enantioselective and nonenantioselective sorption, thermodynamic complexation constants of the single enantiomers with Lipodex E could be determined. The difference in Gibbs free energy -∆E2,E1(∆G) of the complexation of the enantiomers of halodiether B with pure Lipodex E was determined at 30 °C by TSMR and SPR to be 5.7 or 5.9 kJ/mol, respectively, agreeing well with that determined by cGC, i.e., 5.7 kJ/mol at 30 °C.
representative chiral host-guest systems is therefore warranted. To quantify the enantioselective complexation between chiral anesthetics and modifed cyclodextrins, the thermodynamic constants of enantioselectivity were previously determined by capillary gas chromatography (cGC). Indeed, chiral inhalation anesthetics can efficiently be separated into enantiomers with large chiral separation factors R on modified cyclodextrins employed as chiral stationary phase (CSP) by cGC. In cGC, the separation of enantiomers relies on a large number of theoretical plates of the enantioselective capillary column and it is preferentially performed in diluted systems; whereby the chiral host, e.g., the modified cyclodextrin, is diluted in a polymer, e.g., a nonpolar polysiloxane. Since the chiral separation factor R is dependent on the host concentration,2 R does not represent a reliable criterion for enantioselectivity.3 Enantioselectivity is instead related to the difference of the Gibbs free energy between the first eluted enantiomer E1 and the second eluted enantiomer E2, -∆E2,E1(∆G). Adopting the concept of the retention increment R′, which separates the nonenantioselective contribution to retention arising from the achiral polysiloxane solvent and the enantioselective contribution to retention arising from the cyclodextrin host, the enantioselectivity is accessible as follows:
Live processes involving chiral molecules are almost exclusively governed by enantioselective recognition. Thus, for chiral drugs, the relationship between biological activity and stereochemistry is well established. Even for simple chiral inhalation anesthetics, administered as racemic synthetic mixtures, a difference of the narcotic action of the individual enantiomers has been discussed.1 Studies on the enantioselective complexation in
R′E1 and R′E2 are the retention increments of the enantiomers E1 and E2 and rE1 and rE2 are the relative retention data. With r and r0 defined as,
* Authors to whom correspondence should be addressed. (V.S.) E-mail:
[email protected]. Fax: ++49 (0)7071-29 5538. Phone: +++49 (0)7071-29 76257. (G.G.) E-mail:
[email protected]. Fax: ++49 (0)7071-29 5960. Phone: +++49 (0)7071-29 76927. † Institute of Physical and Theoretical Chemistry. ‡ Institute of Organic Chemistry. (1) Franks, N. P.; Lieb, W. R. Nature 1994, 367, 607-613. 10.1021/ac015689k CCC: $22.00 Published on Web 05/17/2002
© 2002 American Chemical Society
R′E2 rE2 - r0 -∆E2,E1∆(G) ) RT ln ) RT ln R′E1 r - r0
(1)
E1
r)
t′ t′*
r0 )
t′0 t′0*
(2)
r is the relative retention of the guest with respect to an inert reference compound on the reactor column containing the host in the polysiloxane solvent, and r0 is the relative retention of the guest with respect to the same reference compound on a reference (2) Jung, M.; Schmalzing, D.; Schurig, V. J. Chromatogr. 1991, 552, 43-57. (3) Juza, M.; Schurig, V. J. Chromatogr., A 1997, 757, 119-135.
Analytical Chemistry, Vol. 74, No. 13, July 1, 2002 3005
column containing the pure polysiloxane solvent. t′ are adjusted retention times. Theory and practice of chemical sensors have reached a high state of development in recent years. Modern sensor technologies are accurate and straightforward tools in analytical chemistry. Recently, enantioselective sensor systems were described for the discrimination of enantiomers requiring no separation in consecutive plates as in cGC but only one single sorption/desorption step. Although they are not as robust as cGC, they are suited for the fast detection and identification of chiral compounds.4 They also can be used for the determination of thermodynamic data of enantioselectivity and even provide information about the mechanism of the host-guest process.5 Using statistically oriented modified cyclodextrins on surfaces, e.g., octakis(3-O-butanoyl-2,6-di-O-n-pentyl)-γ-cyclodextrin (Lipodex E),6,7 thickness shear mode resonators (TSMRs), also known as bulk acoustic wave sensors (BAWs), have already been used for the enantioselective detection of the chiral inhalation anesthetics enflurane, isoflurane, and desflurane at low detection limits.8-9 The stabilization enthalpies of the complexation between different inhalation anesthetics and a modified β-cyclodextrin were calculated and compared to sensor effects,10 and a model for the differentiation between enantioselective and nonenantioselective sorption by cyclodextrins was established.8,11 In this model, the enantioselective contribution to the frequency change ∆f is given by a Langmuir function for each single enantiomer:
K1c ∆fenantioselective ) A 1 + K1c
(3)
K1 determines the initial curvature of the sensor response. From a kinetic point of view, it can be interpreted as the ratio of the kinetic constants of the sorption and desorption process of the guest on a recognition site of the host K1 ) ksor/kdes. The constant A corresponds to the sensor signal at complete coverage of all available sites of the host. The nonenantioselective sorption is given according to Henry’s law:
∆fnonenantioselective ) K2c
(4)
and is supposed to occur at the side chains of the cyclodextrin. The overall sensor response is given by
∆fsum ) ∆fenantioselective + ∆fnonenantioselective
(5)
Cyclodextrin-coated surface acoustic wave (SAW) sensors have (4) Fietzek, C.; Hermle, T.; Rosenstiel, W.; Schurig, V. Fresenius J. Anal. Chem. 2001, 371, 58-63. (5) Bodenho ¨fer, K.; Hierlemann, A.; Seemann, J.; Gauglitz, G.; Christian, B.; Koppenho ¨fer, B.; Go ¨pel, W. Anal. Chem. 1997, 69, 3058-3068. (6) Ko ¨nig, W. A.; Krebber, R.; Mischnick, P. J. J. High Resolut. Chromatogr. 1989, 12, 732-738. (7) Schurig, V.; Grosenick, H. J. Chromatogr., A 1994, 666, 617-625. (8) Bodenho ¨fer, K.; Hierlemann, A.; Juza, M.; Schurig, V.; Go ¨pel, W. Anal. Chem. 1997, 69, 4017-4031. (9) Dickert, F. L.; Geiger, U.; Weber, K. Fresenius J. Anal. Chem. 1999, 364, 128-132. (10) Dickert, F. L.; Reif, H.; Stathopulos, H. J. Mol. Model. 1996, 2, 210-416.
3006
Analytical Chemistry, Vol. 74, No. 13, July 1, 2002
also been used for enantioselective recognition by Hierlemann et al.12 The enantiomers of methyl-2-chloropropionate have been measured at high vapor concentrations. Therefore, the true enantioselective complexation at infinite dilution of the guest cannot be revealed. Since the enantioselective detection by sensors is concentration-dependent, the inherent difference of the complexation constant should be examined over a broad range of concentrations as the difference of the enantiomers at one concentration describes the system insufficiently. In regard to sensors based on surface plasmon resonance (SPR), as far as we know only monolayers of modified cyclodextrins have been used as sensor coatings.13 For reflectometric interference spectroscopy (RIfS), no enantioselective detection of vapors using cyclodextrins has been performed up to now. In this contribution, we investigated the use of Lipodex E for the enantioselective detection of chiral haloethers over a broad vapor concentration range. Therefore, we used sensors with different transduction principles including TSMR, SAW, SPR, and RIfS. The results were then compared to that obtained from cGC measurements. The comparison of the different sensor methods should provide the validation of the determined constants and therefore of the model used for the description of the host-guest interaction. The advantages and limitations of each sensor method will be discussed. For such a comparison, a substance with a high chiral separation factor R in cGC was considered. 1,1,1,3,3-Pentafluoro2-(fluoromethoxy)-3-methoxypropane (halodiether B) is a degradation product of the inhalation anesthetic sevoflurane (1,1,1,3,3,3hexafluoro-2-(fluoromethoxy)propane),14,15 which is reactive in the presence of alkaline carbon dioxide absorbents.16,17 The separation of the enantiomers of halodiether B (as racemic guest) by cGC on the modified cyclodextrin heptakis(2,3-di-O-acetyl-6-O-tert-butyldimethylsilyl)-β-cyclodextrin (as chiral host) showed a separation factor of R ) 4.1 at 30 °C.18 Recently, an even larger separation factor R ) 9.7 at 30 °C was detected by using Lipodex E. Thus, this highly enantioselective host-guest system will be probed by a multitude of sensor devices. A theoretical description of the fundamentals of the applied chemical sensor devices is given in the following section. FUNDAMENTALS ON THE APPLIED CHEMICAL SENSING DEVICES Measuring the mass uptake of a sensitive coating upon exposure to vapors is a straightforward concept in chemical sensing. Acoustic wave (AW) sensors are a prominent class of mass-sensing devices. They are widely applied and well characterized and have been the topic of books and for gas- and liquid(11) Michalke, A.; Janshoff, A.; Steinem, C.; Henke, C.; Sieber, M.; Galla, H.-J. Anal. Chem. 1999, 71, 2528-2533. (12) Hierlemann, A.; Ricco, A. J.; Bodenho ¨fer, K.; Go ¨pel, W. Anal. Chem. 1999, 71, 3022-3035. (13) Weisser, M.; Nelles, G.; Wenz, G.; Mittler-Neher, S. Sens. Actuators, B 1997, 38/39, 58-67. (14) Frink, E. J.; Malan, T. P.; Morgan, S. E.; Brown, E. A.; Malcomson, M.; Brown, B. R. Anesthesiology 1994, 80, 71-76. (15) Bito, H.; Ikeda, K. Anesthesiology 1994, 80, 71-76. (16) Sturm, D. P.; Johnson, B. H.; Eger, I. I. Anesthesiology 1987, 67, 779-781. (17) Morio, M.; Fujii, K.; Satoh, N.; Imai, M.; Kawakami, U.; Mizuno, T.; Kawai, Y.; Ogasawara, Y.; Tamura T.; Negishi A.; Kumagai, Y.; Kawai, T. Anesthesiology 1992, 77, 1155-1164. (18) Schmidt, R.; Roeder, M.; Oeckler, O.; Simon, A.; Schurig, V. Chirality 2000, 12, 751-755.
phase sensing.19-23 Usually AW sensors consist of a piezoelectric substrate with thin films or interdigitated metal electrodes used to convert electrical energy to mechanical energy in the form of an AW. The key feature of AW sensors is that the frequency and the amplitude of the AW is affected by a mass changes of the system. In this contribution, we use SAWs generated by an interdigitated resonator structure as well as bulk acoustic waves generated by a TSMR. As shown by Sauerbrey24 for TSMRs and Wohltjen25 for SAWs, the vibrating frequency of a quartz crystal decreases upon the mass uptake of the sensitive layer. The frequency shift ∆f is to a first approximation proportional to the mass ∆m deposited onto the surface:
∆f ) -Cf 02∆m/S
(6)
where C denotes a constant, f0 the fundamental frequency, and S the area of the sensing surface. This so-called Sauerbrey equation predicts the sensor response ∆f to be proportional to the fundamental frequency by a power of 2. The higher the fundamental frequency of the device, the larger the sensor response to be expected. Although SAW sensors are able to operate at higher device frequencies compared to TSMRs, they are strongly affected by changes in the physical properties of the sensing layer.26,27 To exclude the influence of additional effects, special care has to be made in the choice of the sensing material, especially by using thin-layer thickness and low-viscosity material. With SPR, the change of the refractive index of the sensitive layer is determined by measuring the shift of the resonance wavelength or the shift of the resonance angle of the incident light. The resonance condition for the surface plasmon is given by eq 7,28 where 1 and 〈〉 are the dielectric constants of the prism
sin Θx1(λ) )
x
2,re(λ)〈〉(λ)
2,re(λ) + 〈〉(λ)
(7)
and of the sensitive layer, 2,re is the real part of the dielectric constant of a metal layer, e.g., silver or gold, Θ is the angle of the incident light, and λ is the wavelength of irradiation. Since all dielectric constants depend on the wavelength of the incident light, no simple relation between the resonance wavelength and the dielectric constant of the sensitive layer exists. At a constant angle, the resonance wavelength always shifts to higher wavelength on an increasing dielectric constant of the sensitive layer.29 However, for a fixed angle of the incident light, a fixed prism, and metal, the relation between the resonance wavelength (19) Grate, J. W. Chem. Rev. 2000, 100, 2627-2648. (20) Grate, J. W.; Frye, G. C. Acoustic wave sensors. In Sensors Update; Baltes, H., Go ¨pel, W., Hesse, J., Eds.; VCH: Weinheim, 1996; Vol. 2. (21) Martin, S. J.; Frye, G. C.; Senturia, S. D. Anal. Chem. 1994, 66, 22012219. (22) Mecea, V. M. Sens. Actuators, A 1994, 40, 1-27. (23) Grate, J. W.; Martin, S. J.; White, M. W. Anal. Chem. 1993, 65, 987A. (24) Sauerbrey, G. Z. Phys. 1959, 155, 206. (25) Ballantine, D. S.; Wohltjen, H. Anal. Chem. 1989, 61, 706. (26) Bodenho ¨fer, K.; Hierlemann, A.; Noetzel, G.; Weimar, U.; Go ¨pel. W. Anal. Chem. 1996, 68, 2210-2218. (27) Ricco, A. J.; Martin, S. J. Thin Solid Films 1991, 206, 94-101. (28) Raether, H. Phys. Thin Films 1977, 9, 145-261. (29) Homola, J. Sens. Actuators, B 1997, 41, 207-211.
and the dielectric constant of the sensitive layer can be calculated by numerical solvation of eq 7 or of Fresnels equations.30 For isotropic, flexible, sensitive layers, the volume fraction of the analyte within the sensitive layer can be calculated by using Bruggemanns equation,31
3 - 〈〉 4 - 〈〉 + Φ4 0 ) Φ3 3 + 2〈〉 4 + 2〈〉
(8)
where 3, 4, and 〈〉 are the dielectric constants of the pure sensitive layer, the analyte, and the mixture, respectively. Φ3 and Φ4 are the volume fractions. With RIfS, the volume fraction of the analyte in the sensitive layer is determined directly. The fundamentals of RIfS are based on interference effects at thin transparent films. Upon passing a transparent sensitive layer, a light beam is reflected in part at each of the interfaces (air/sensitive layer and sensitive layer/support). As the two reflected beams travel different optical paths, a phase shift is introduced. The interference effects can be observed for short coherent (white) light sources. A wavelength-dependent modulation of reflected light intensity due to constructive and destructive interference follows. In the case of perpendicular incidence of light, the reflectance R of a thin transparent layer as a function of the wavelength is given by
(4πnd λ )
R ) R1 + R2 + 2xR1R2 cos
(9)
where R1 and R2 denote the Fresnel reflectances at the film interfaces, n is the refractive index of the sensitive layer, d is the physical thickness of the sensitive layer, and λ the wavelength of the incident light. By sorption of guest molecules into the sensitive layer, the optical thickness of the layer is changed, causing a shift in the interference spectrum. Consequently, changes in sensitive layer thickness can be determined from the interference spectra with high resolution. EXPERIMENTAL SECTION Materials. Sevoflurane and racemic isoflurane were kindly provided by the local university clinics of Tu¨bingen. The refractive index at 30 °C was determined to nD ) 1.29. The racemic mixture of halodiether B was prepared according to a modified method18 described by Huang et al.33 The refractive index at 30 °C was determined to nD ) 1.325. The polysiloxane PS255 (polydimethyl(0.5-1%)-methylvinylsiloxane copolymer) was obtained from ABCR (Karlsruhe, Germany). Lipodex E6 was synthesized as described in ref 7. Chromosorb P AW DMCS 80/100 mesh was purchased from Macherey-Nagel (Du ¨ ren, Germany). All other chemicals were obtained from Fluka (Deisenhofen, Germany). The solvents were dried according to standard procedures. The deactivated fused-silica capillaries (0.25-mm i.d.) were purchased from Ziemer GmbH (Mannheim, Germany). (30) Kraus, G.; Gauglitz, G. Fresenius J. Anal. Chem. 1992, 344, 153-157. (31) Bruggemann, D. A. G. Ann. Phys. 1935, 24, 636-665. (32) Huang, C. G.; Rozov, L. A.; Halpern, D. F.; Vernice, G. G. J. Org. Chem. 1993, 58, 7382-7387. (33) Huang, C.; Venturella, V. S.; Cholli, A. L.; Venutolo, F. M.; Silbermann, A. T.; Vernice, G. G. J. Fluorine Chem. 1989, 45, 239-253.
Analytical Chemistry, Vol. 74, No. 13, July 1, 2002
3007
Isolation of the Halodiether B Enantiomers by Preparative GC. A multidimensional preparative GC AMPG 60 (Gerstel, Germany) described by Schomburg et al.34 was used. It consists of a precolumn (1 m, 14-mm i.d., Volaspher A 4 (Merck, Darmstadt, Germany), 60/80 mesh coated with polysiloxane 10% SE 30), and a main column (1 m, 18-mm i.d.;18 Chromosorb P AW DMCS 80/100 mesh, coated with 19.1% heptakis(2,3,-di-Oacetyl-6-O-tert-butyldimathylsilyl)-β-cyclodextrin) diluted by 15.2% in PS 86). The temperature was 70 °C, carrier gas N2, and flow 240 mL/min, and the operational mode was peak-cutting and backflushing. Halodiether B was dissolved in diethyl ether (1:5) and cooled to -20 °C prior to injection. Sample-volume/single-cycle 250 µL, with separation time for a single cycle 55 min. Number of cycles, 21. With this procedure, 316 mg of (R)-(-)-halodiether B (chemical purity 99.9%, enantiomeric excess 99.9%) and 261 mg of (S)-(+)-halodiether B (chemical purity 99.6%, enantiomeric excess 97.9%) were obtained. Caution. Inhalation of these anesthetics causes immediate unconsciousness and dizziness. Therefore, all the experiments have to be performed in a hood or in closed gastight setups. Gas Chromatography. The fused-silica capillary columns coated with chiral selectors ((hexakis-, heptakis-, or octakis(3-Obutanoyl-2,6-di-O-pentyl)-γ-cyclodextrin dissolved in PS255) were prepared by the static method.35 An HP 5890A gas chromatograph (Agilent, Waldbronn, Germany) equipped with an FID (250 °C) was used. Thickness Shear Mode Resonators and Surface Acoustic Wave Resonators. As TSMR transducers, discrete piezoelectric quartz crystals (AT-cut) with gold electrodes operating at a fundamental frequency of f0 ) 30 MHz (KVG, Quartz Crystal Technology GmbH) were used. The frequency outputs of the TSMRs were recorded every 30 s with 0.1-Hz resolution. As SAW transducers, devices of the type E062 G/H (Siemens AG) operating at frequencies between 430 and 437 MHz were used. An uncoated SAW inside the measurement chamber was used to compensate for temperature effects and pressure changes. The first monitored frequency value of each device was set to zero; hence, all frequency differences assessed thereafter refer to this first value. SAW and TSMR sensor signals were recorded by using the open bench system MOSES II (Lennartz Elektronik GmbH, Tu¨bingen, Germany), which is capable of measuring different sensor types simultaneously. Up to eight TSMR and seven SAW sensors were measured at one time. Typical response times of the sensors were observed in the order of seconds during alternating exposures to air and different vapor concentrations. Surface Plasmon Resonance. SPR measurements were achieved using a system based on the Kretschmann configuration.36 The experimental setup is shown in Figure 1a. TM-polarized polychromatic light (5 V/5 W krypton with integrated reflector, Welch Allyn, New York, NY) is focused on the prism at a fixed angle (61°). Surface plasmons are excited at the prism’s back surface. The modulated output light is coupled into a multimode fiber and detected with a diode array spectrometer (Zeiss MCS, (34) Schomburg, G.; Ko ¨tter, H.; Stoffels, D.; Reissig, W. Chromatographia 1984, 19, 382-390. (35) Grob, K. Making and manipulating capillary columns for gas chromatography; Dr Alfred Hu ¨ thig Verlag: Heidelberg, 1986; pp 156-176. (36) Kretschmann, E.; Raether, H. Z. Naturforsch. 1968, 23A, 2135-2136.
3008 Analytical Chemistry, Vol. 74, No. 13, July 1, 2002
Figure 1. Measurement setups for (a) SPR and (b) RIfS measurements.
Jena, Germany). Because the surface plasmon wave is only excited by the TM mode, the TE mode is used as a reference signal. The substrate was prepared by evaporating gold onto the glass prism (halfcylindric, ground 20 × 40 mm, SF13, Schott) using a vacuum evaporation system (Pfeiffer Vacuum GmbH, Wetzlar, Germany). A thin layer of titanium (∼3 nm) was placed on the glas prism to help promote adhesion of the gold layer. Deposition of gold was performed at high vacuum (10-7 mbar) and at a rate of about 0.8-1.5 nm s-1. Both gold and titanium thicknesses were monitored by a crystal oscillator. Approximately 50 nm of gold was deposited onto the glass. The dielectric constants of gold were determined by spectral ellipsometry (ES4G, SOPRA). The Cauchy parameters of the prism are A ) 1.702 52, B ) 1.3 × 104 nm2, and C ) 2.2 × 108 nm.4 With these parameters, the change in the refractive index of the sensitive layer was determined from the change in the resonance wavelength using Fresnel equations. Reflectometric Interference Spectroscopy. A sensor array configuration was used as demonstrated in Figure 1b. It consists of an infrared filtered tungsten white light source (100 W) and a diode array spectrometer (Zeiss MCS) with both connected by a fiber-optic coupler bundle (diameter, 1-mm poly(methyl methacrylate) (PMMA), Microparts, Dortmund, Germany). The one end of the bundle faces the lamp to collect homogeneous light. The fibers transmit the radiation to the glass substrate coated with the sensitive layer. Radiation reflected from the interfaces of this thin sensitive layer exhibits wavelength-dependent interference. The reflected radiation is transmitted via the coupler to an optical multiplexer (Laser Components, Olching, Germany), which is directly connected to the spectrometer. Data recording and evaluation were done with software developed in our laboratory.30 This software calculates the change of the optical thickness of the sensitive layer for each sensor element within each measurement cycle using the interference fringe by evaluation of prominent data points of the interference spectrum. Coating Procedure. All the TSMR and SAW sensors were prepared by airbrush coating. The coating materials were dis-
solved in dichloromethane (∼1 mg/mL) and sprayed onto the cleaned surfaces using synthetic air as the carrier gas. During the coating process, the frequency change of the resonators was monitored. The coating process was stopped for the TSMR as soon as a shift of 35 kHz was reached on each side. An overall frequency shift of 70 kHz corresponds to a layer thickness of at least 160 nm per side, assuming a homogeneous layer and a density of 1.2 g/mol. For SAW, the coating process was stopped as soon as a damping of 9 dB was reached. The RIfS sensors were prepared by spin-coating. Lipodex E was dissolved for 10 and 20% by mass solutions in toluene. A 20µL aliquot of the solution was coated onto the cleaned surface by 5000 rpm and 40 s. Layer thickness was typically between 250 and 300 nm with little homogeneity. The polysiloxane PS255 was dissolved in toluene (5%) and 20 µL of the solution was coated onto the cleaned surface by 5000 rpm and 40 s. For SPR, the polysiloxane layers were prepared similarly. The polysiloxane PS255 was dissolved in toluene (5%), and 20 µL of the solution was coated onto the cleaned surface by 3000 rpm and 40 s. Since homogeneous Lipodex E layers could not be achieved by spray-coating or by spin-coating, the SPR layers were formed by dip-coating of a 2% solution of Lipodex E in toluene. Although the surface is not flat, the gold layer is totally covered and the thickness of the Lipodex E layers extends more than 1 µm. Therefore, the evanescent field does not extend beyond the layer. Gas Mixing and Setup. The test gases were generated from temperature-controlled (T ) 253-293 K) vaporizers with the concentration in the gas phase given by Antoine’s law. Defined concentrations were adjusted by using a gas mixing apparatus.37 The saturation pressure of halodiether B was determined to be 53 ( 2.6 Pa at -25 °C, 294 ( 3.6 Pa at -5 °C, and 1184 ( 50 Pa at 10 °C by evaporating a known amount of the liquid as described by Bodenho¨fer et al.38 Defined vapor concentrations were obtained by using nitrogen as carrier gas and by dilution with computer-driven mass flow controllers. All vapors were mixed before entering the measurement chambers. The chambers remained at a constant temperature of 303 K. The total gas flow rate (analyte gas and nitrogen) to the SPR and RIfS sensors was adjusted to 300 mL/min at a total pressure of 105 Pa. Due to the miniaturized tubing and measurement chambers with volumes of 2 (TSMR) and 3 mL (SAW) each, the MOSES II system was set up in a bypass arrangement with a flow of 25 mL/min behind the optical sensors. RESULTS AND DISCUSSION Separation of Enantiomers by cGC. Table 1 shows the chiral separation factors R of halodiether B for hexakis-, heptakis-, and octakis(3-O-butanoyl-2,6-di-O-n-pentyl)cyclodextrin as 20% (w/w) in PS255 determined by cGC. With the R-cyclodextrin, no separation was achieved whereas the β-cyclodextrin produced a relatively high separation factor. For the γ-cyclodextrin (Lipodex E) we determined the highest known chiral separation factor in (37) Kraus, G.; Gauglitz, G. Chem. Intell. Lab. Syst. 1995, 30, 211. (38) Bodenho ¨fer, K.; Hierlemann, A.; Schlunk, R.; Go ¨pel, W. Sens. Actuators, B 1997, 45, 259-264.
Table 1. Chiral Separation Factors r of Halodiether B for Hexakis-, Heptakis-, and Octakis(3-O-butanoyl-2,6-di-O-n-pentyl)cyclodextrin 20% in PS255 T (°C)
R (R-CD)
R (β-CD)
R (γ-CD)
30 40 50
1.0 1.0 1.0
2.1 1.6 1.4
9.7 7.7 6.0
Figure 2. Gas chromatographic separation of enantiomers of halodiether B at 30 °C using Lipodex E.39
gas chromatography of R30 °C ) 9,7.39 The separation is shown in Figure 2. All further sensor measurements of halodiether B have therefore been performed using Lipodex E. Comparison of Information Provided by Different Transducers. To determine all model constants for the interaction of the enantiomers of halodiether B with Lipodex E, the calibration curves were measured over a broad range of vapor concentrations. A comparison of information provided by different transducers and the determination of the stoichiometric ratio of the hostguest complexation can already be made by using the racemic mixture of halodiether B. In Figure 3, the calibration curves measured by TSMR and SPR at 30 °C are shown for the racemic mixture of halodiether B on sensors with PS255 and Lipodex E coatings, respectively. The signals were acquired while the devices were exposed to an alternating sequence of synthetic air and various concentrations of halodiether B in random series. Each concentration was measured three times, and the standard deviation is given in the error bars. Regarding the SPR signals, a decrease of the refractive index is observed. This can be explained if there are no fixed cavities inside the Lipodex E layers. In this case, as for the polysiloxane PS255, halodiether B replaces the Lipodex E within the evanescent field and because its refractive index is smaller a decrease of the refractive index occurs with higher concentrations of halodiether B. For the TSMR sensors, a mass uptake results in a frequency decrease of the fundamental mode. A frequency decrease was also observed for the generated mode of the SAW. For RIfS, the analyte uptake results in a swelling of the sensitive layer. In this case, the sensor signal shows the relative change of the optical path length ∆(nd)/(nd)0. A different behavior for the Lipodex E-coated sensors as compared to the achiral PS255-coated sensors was observed for (39) Schmidt, R. Enantiomeranalytik chiraler Inhalationsana¨sthetika und ihrer Zersetzungsprodukte mittels GC, Dissertation, Eberhard-Karls-Universita¨t Tu ¨ bingen, 2001.
Analytical Chemistry, Vol. 74, No. 13, July 1, 2002
3009
Table 2. Model Parameters for the Racemic Mixture of Halodiether B and for the Racemic Mixture of Isoflurane Obtained by SPR, TSMR, RIfS, and SAW at 30 °C SPR
A
K1,racemate (L/µg)
K2 (L/µg)
KPS255 (L/µg)
A/K2 (µg/L)
halodiether B isoflurane
2.33 × 10-3 ( 0.05 × 10-3 3.56 × 10-3 ( 0.07 × 10-3
9.5 × 10-4 ( 0.4 × 10-4 1.5 × 10-5 ( 0.1 × 10-5
5.5 × 10-8 ( 0.4 × 10-8 2.2 × 10-8 ( 0.03 × 10-8
9.6 × 10-9 ( 0.09 × 10-9 3.8 × 10-9 ( 0.05 × 10-9
42400 ( 3200 163000
K2 (Hz‚L/µg)
KPS255 (Hz‚L/µg)
TSMR halodiether B isoflurane
A (Hz) 2510 ( 80 2370 ( 30
RIfS halodiether B
SAW halodiether B
K1,racemate (L/µg) 10-4
1.6 ×
10-2
( 0.6 × 9.4 × 1.5 × 10-5 ( 0.2 × 10-5
( 0.12 × A (Hz)
66200 ( 3400
10-2
10-2
8.3 ×
10-4
( 1.3 ×
10-4
K1,racemate (L/µg) 4.5 ×
10-4
( 0.4 ×
10-4
Figure 3. Calibration curves at 30 °C for the racemic mixture of halodiether B with PS255 (points) and Lipodex E (squares) as sensor coatings measured by (a) TSMR and (b) SPR.
all transducer types. Signals of the Lipodex E-coated sensors were more than 1 order of magnitude higher than those of the PS255coated sensors, and the isotherm obtained by the chiral coating showed a pronounced nonlinear behavior. The latter indicates a defined complexation site as observed by cGC. For further evaluation, we therefore applied the model of enantioselective and nonenantioselective complexation (eqs 3-5). The data obtained by fitting the experimental data of Figure 3 as well as the experimental data for SAW and RIfS sensors are listed in Table 2. The complexation constant of the racemic mixture K1R, which determines the curvature of the isotherm, has the same dimension for all transducers and is therefore best suited for a comparison between different transducers. For the SPR and TSMR identical complexation constants of 9.5 × 10-4 ( 0.4 × 10-4 and 9.4 × 10-4 ( 0.6 × 10-4 L/µg are found. Regarding the constants A and K2, no direct comparison can be made because the units are not the same for all transducers. However, if the ratios of A and K2 are calculated, the different units are canceled out and values of 42 400 ( 3200 and 42 500 ( 3700 µg/L are obtained. In Table 2, the 3010 Analytical Chemistry, Vol. 74, No. 13, July 1, 2002
10-3
5.90 × ( 0.5 × 2.1 × 10-2 ( 0.02 × 10-2
K1,racemate (L/µg)
A 10-2
10-4
4.4 × ( 0.3 × 4.7 × 10-3 ( 0.2 × 10-3
K2 (L/µg) 4.7 ×
10-7
( 0.7 ×
A/K2 (µg/L)
10-3
KPS255 (L/µg) 10-7
5.5 ×
10-8
( 0.1 ×
42500 ( 3700 127000
A/K2 (µg/L) 10-8
34300 ( 5500
K2 (Hz‚L/µg)
KPS255 (Hz‚L/µg)
A/K2 (µg/L)
2.7 ( 0.2
0.7 ( 0.02
24600 ( 2000
constants for the racemic mixture of the inhalation anesthetic isoflurane are shown, which shows the proportionality of the signals for TSMR and SPR. Therefore, the TSMR and SPR results prove that the change in refractive index is proportional to the mass change, which itself is proportional to the concentration of halodiether B in the sensitive layer. The same model of enantioselective and nonenantioselective sorption is valid for SPR and for TSMR. It is known that Bruggemann’s eq 8 is valid for the nonenantioselective sorption, and since enantioselective and nonenantioselective sorption show the same proportionality between TSMR and SPR signals, Bruggemann’s equation should be valid for the enantioselective sorption, too. The complexation constant K1R, A, and the constant K2 can be determined. The accuracy of the measurement is in the same range for SPR and TSMR. Smaller complexation constants K1R and smaller ratios of A over K2 are found for SAW and the RIfS sensors. Since the complexation constants K1 are expected to be the same for all sensor types, the model of the Langmuir- and Henry-type sorption is not valid for these transducers. This difference for the SAW is explained by applying much thinner layers of the pure Lipodex E than for the other sensor types. Observation of the sensor surface by a conventional light microscope revealed uncoated surface areas. This is probably caused by the strong cohesion of the applied Lipodex E. The incomplete coverage of the transducer surface by Lipodex E might result in additional achiral contributions to the sensor signal and hence in an overall reduction of the experimental SAW separation factor. Regarding the RIfS measurements, the sensitive layers had no planar surfaces, which in addition changed during the measurements. This resulted in small intensities of the reflected light and drift of the baseline. However, the surface structure is not influencing the TSMR measurement or the SPR measurements, where the evanescent field does not extend beyond the sensitive layer. Determination of the Stoichiometric Ratio of the HostGuest Complexation. The molecular numbers of isoflurane and the molecular numbers of halodiether B, at complete coverage of all complexation sites of Lipodex E, can be determined by TSMR and SPR. A stoichiometric number for the host-guest complex
Table 3. Sensitivities of Achiral TSMR Polymer Sensors for Racemic Halodiether B and the Single Enantiomers K2 (S)-halodiether B(Hz‚L/µg) PS255 PEUT
10-3
K2 racemic halodiether B (Hz‚L/µg)
10-3
10-3 (
( 0.2 × 4.2 × 5.1 × 10-2 ( 0.3 × 10-2
10-3
4.4 × 0.3 × 5.4 × 10-2 ( 0.2 × 10-2
K2 (R)-halodiether B (Hz‚L/µg) 4.3 × 10-3 ( 0.2 × 10-3 5.0 × 10-2 ( 0.2 × 10-2
can be given. For the TSMR, the stoichiometry x of the hostguest complex can be determined by using the following equation:
Xstoichiometric ) AmLipodex E/∆fLipodex EMhalodiether B (10) where ∆fLipodex E denotes the frequency decrease after the coating procedure. We measured this to be 70 kHz. A denotes the frequency change caused by enantioselective sorbed guest (see Table 2), respectively. M denotes the molecular masses of the compounds (MLipodex E ) 2980 g/mol, Mhalodiether B ) 212.1 g/mol, Misoflurane ) 184.5 g/mol). A value of 0.50 is obtained for halodiether B and 0.55 for isoflurane. For SPR, the refractive indices of the sensitive layer and of the guests must be determined. The refractive index of Lipodex E is n30 °C ) 1.42 ( 0.01 at the resonance wavelength. The refractive indices of isoflurane and halodiether B are nD,isoflurane, 30 °C ) 1.29 and nD,halodiether B, 30°C ) 1.325. If the dispersion of the refractive index is neglected, the volume fractions for the enantioselective sorbed guest Φ4 can be calculated using Bruggemann’s eq 8 and are determined to be 2.75 × 10-2 ( 2 × 10-3 for isoflurane and 2.45 × 10-2 ( 3 × 10-3 for halodiether B. With Fisoflurane ) 1.5 g/cm3 and the estimated densities for halodiether B Fhalodiether B ) 1.5 g/cm3 and for Lipodex E FLipodex E e 1.3 g/cm3 with
Xstoichiometric ) Fhalodiether BMLipodex EΦ4/FLipodex EMhalodiether B (11) the mole fraction can be calculated to g0.51 for isoflurane and g0.40 for halodiether B, which indicates as the ratio determined by TSMR that about half of the host molecules are involved in the process of complexation. These results agree with observations made with other cyclodextrin systems, where stoichiometries between 1 and 0.5 have been determined.4,8 Determination of Enantiomers by Sensors. Measurements with the pure enantiomers were performed below concentrations of 1000 µg/L. The dosing of the halodiether B was checked by TSMR reference sensors coated with the achiral polyetherurethane (PEUT) and the achiral polysiloxane (PS255). These sensors were investigated in parallel with the chiral sensors. As expected for achiral coatings, the same values were found for the two enantiomers. The isotherms showed linear behavior. The same slopes were found for both enantiomers within the experimental errors, listed in Table 3. In Figure 4, the response of the SPR and SAW sensors upon exposure to the single enantiomers of halodiether B are shown. At the same analyte concentrations, signals from the second eluted S-enantiomer in cGC are much higher than for the R-enantiomer for both transducers. Analogous to cGC, an enantioselectivity factor R can be determined by dividing the signal heights. For a concentration of 20 µg/L, the value R for SPR is 9.6 ( 0.7 and 8.3 ( 1.6 for TSMR. These values decrease at higher concentration,
Figure 4. Calibration curves of single enantiomers of halodiether B for (a) SAW and (b) SPR with Lipodex E at 30 °C.
because more complexation sites are occupied by guest molecules. For low concentrations, the R values obtained are the same as for the cGC measurement within experimental errors. Although the SAW experiments result in much higher signals for the S-enantiomer, the separation factors R of the SAW sensors only show values between 5 and 7. As already mentioned above, another effect resulting from the unoccupied transducer surface occurs for both enantiomers. However, regarding Figure 5, it can be seen that due to the relatively thick sensor coating on the SPR sensors the response time is slow. Exposure times of 60 min were chosen in order to obtain equilibrium signals for the SPR signals and to determine thermodynamic constants. The response time is much faster for the SAW sensors. It is concluded that for practical sensoric determination of the enantiomers of halodiether B, the SAW sensor with a response time of less than 2 min is most suitable. However, using SPR and TSMR sensors, an exact determination of the complexation constants of the single enantiomers is possible. The different complexation constants were obtained by curvature fitting of the sensor values obtained at the small concentration range, depicted in Figure 4. During the fitting procedure, A and K2 of the racemic mixture were kept constant because they are the same for both enantiomers. The obtained complexation constants are listed in Table 3. By using these values, differences in Gibbs energy -∆E2,E1(∆G) can be determined by using eq 9. Contrary to cGC, no reference to subtract nonenantioselective interactions is needed.
-∆E2,E1∆(G) ) RT ln(K1,E2/K1,E1)
(12)
According to Table 2, all transducers showed that the nonenantioselective interaction of halodiether B with the Lipodex E is higher than the interaction with the polysiloxane PS255. Therefore, Analytical Chemistry, Vol. 74, No. 13, July 1, 2002
3011
Table 4. Model Parameters for the Single Enantiomers of Halodiether B Obtained by SPR and TSMR at 30 °C
SPR halodiether B TSMR halodiether B
K1,racemate (L/µg)
K1, (R)-halodiether B (L/µg)
K1, (S)-halodiether B (L/µg)
K1S/KR
∆E2,E1(∆G) (kJ/mol)
9.5 × 10-4 ( 0.4 × 10-4 9.4 × 10-4 ( 0.6 × 10-4
1.66 × 10-4 ( 0.04 × 10-4 1.50 × 10-4 ( 0.08 × 10-4
1.77 × 10-3 ( 0.04 × 10-3 1.46 × 10-3 ( 0.05 × 10-3
10.6 ( 0.3 9.7 ( 0.6
-5.9 ( 0.1 -5.7 ( 0.2
Figure 5. (a) SAW signals and (b) SPR signals with Lipodex E as sensor coating on exposure to the single enantiomers of halodiether B at 30 °C in a concentration range between 0 and 140 µg/L.
even with a reference column, not all of the nonenantioselective interaction can be excluded and the values of the retention increment R’ still include a small amount of nonenantioselective interaction. Therefore, the determination of the difference of Gibbs energy of the two enantiomers by cGC was performed using only 5-10% Lipodex E diluted in polysiloxane to -∆E2,E1(∆G) ) 5.7 kJ/mol.39 In this case, the additional nonenantioselective interaction can be neglected. The comparison of the cGC and sensor measurements as shown in Table 4 confirms this result. Compared to cGC, -∆E2,E1(∆G) is the same for SPR and TSMR sensor within experimental errors. CONCLUSION With Lipodex E, halodiether B could be separated at 30 °C with a separation factor of R ) 9.7, which is the highest separation factor found for chiral separations on cyclodextrins by cGC up to now. The good agreement for TSMR and SPR with the results obtained by cGC is explained by the robustness against structural
3012 Analytical Chemistry, Vol. 74, No. 13, July 1, 2002
changes of the surface. For these techniques, the enantioselective complexation has been separated from nonenantioselective sorption by applying a model based on Langmuir- and Henry-type sorption. The same complexation constants of the enantiomers of halodiether B were obtained for SPR and TSMR sensors and allowed the determination of -∆E2,E1∆(G) without use of a reference. The same value for -∆E2,E1∆(G) was determined as with cGC. Chemical sensor measurements of the saturation signals of halodiether B allowed measurements at different vapor concentrations and, hence, the measurements of isotherms. Interpretation of the isotherms suggests that only half of the cyclodextrins hosts are involved in the complexation process. Although SAWs were not useful for the determination of thermodynamic constants, they are important sensors for practical application since the response time is very fast. SAWs could be used for on-line determination of inhalation anesthetics or degradation compounds such as halodiether B. ACKNOWLEDGMENT This work was supported by Deutsche Forschungsgemeinschaft (DFG) in the framework of the research group “Molekulare Mustererkennung mit supramolekularen Strukturen und Polymeren”, the DFG graduate college “Analytical Chemistry” at the University of Tu¨bingen, and the Fonds der Chemischen Industrie. We thank Dr. Rozow, Baxter Healthcare Co., USA, for isoflurane enantiomers and precursor, respectively. Thanks are also due to Dr. D. Belder, D. Stoffels, and W. Reissig, Max Planck Institut fu¨r Kohlenforschung Mu¨lheim/Ruhr, Germany, for experimental help in preparative GC. We gratefully acknowledge valuable technical support by Lennartz Elektronik GmbH, Tu¨bingen, Germany. Received for review November 14, 2001. Accepted February 13, 2002. AC015689K
