Anal. Chem. 1998, 70, 5184-5189
Oxygen Sensing in Nonaqueous Media Using Porous Glass with Covalently Bound Luminescent Ru(II) Complexes Marı´a P. Xavier,† David Garcı´a-Fresnadillo,† Marı´a C. Moreno-Bondi,*,‡ and Guillermo Orellana*,†
Laboratory of Applied Photochemistry, Department of Organic Chemistry and Laboratory of Optical Sensors, Department of Analytical Chemistry, Faculty of Chemistry, Universidad Complutense de Madrid, E-28040 Madrid, Spain
A comparative study of the oxygen-quenching features of novel luminescent Ru(II) complexes, covalently attached to controlled porous glass (CPG), has been performed in methanol, chloroform, toluene, cyclohexane, and n-hexane. The investigated dyes contain 1,10-phenanthroline4,7-bis(phenylsulfonate) (s2d), 5-acetamide-1,10-phenanthroline (acap), and 5-tetradecanamide-1,10-phenanthroline (tdap) coordinated to the Ru(s2d)2 moiety. The immobilization procedure involves stable sulfonamide linkages to amino-derivatized CPG-Glycophase, the loading being a function of indicator molecular size (i.e., highest for the [Ru(s2d)2(acap)]2- dye). Steady-state and time-resolved emission analysis have revealed that oxygen quenching is purely dynamic and occurs on the glass bead surface. Using a continuous-flow system and a conventional spectrofluorometer, a detection limit of 6.2 µM (200 ppb) oxygen in methanol has been achieved and a 2-4-fold one in the other organic solvents tested, with dynamic ranges over two decades. The analytical figures of merit make the fabricated sensing phases suitable for fiber-optic industrial monitors and oxygen transducers for optical biosensing in nonaqueous media. Development of optical sensors for oxygen measurements in nonaqueous samples is a matter of considerable interest, particularly in relation to process control (e.g., petrochemical industry, caprolactame production, synthesis of fine chemicals by photooxidation, etc.). The widely used electrodes for oxygen monitoring in aqueous media cannot be used in organic solvents due to chemical attack to their sensing polymer membrane or interference from permeated solvents. Therefore, expensive paramagnetic oxygen analyzers or solid-state transducers must be used to quantify the analyte in the gas phase above the liquid sample. The importance of such optical sensors not only lies in the determination of oxygen itself but also in being capable of acting as transducers for various biocatalyzed reactions where oxygen is consumed or produced.1,2 The aforementioned versatility offers new possibilities for biosensing technology in the clinical and †
Laboratory of Applied Photochemistry. Laboratory of Optical Sensors. (1) Saini, S.; Hall, G. F.; Downs, M. E. A.; Turner, A. P. F. Anal. Chim. Acta 1991, 249, 1. (2) Wang, J. Talanta 1993, 40, 1397. ‡
5184 Analytical Chemistry, Vol. 70, No. 24, December 15, 1998
environmental fields and extends the number of detectable analytes to include organic species poorly soluble in water (metabolites, phenols, chlorinated biphenyls, pesticides). Molecular oxygen is a well-known quencher of both singlet and triplet electronic excited states. Polycyclic aromatic hydrocarbons,3-5 porphyrins,6 and transition metal complexes7-12 are the most commonly used luminescent indicators for oxygen sensing. Among the latter, the coordination compounds of ruthenium(II) with polyazaaromatic chelating ligands13,14 offer definite advantages such as (photo)stability, large Stokes’ shifts, strong absorption in the blue region of the electromagnetic spectra, and significantly long emission lifetimes (0.1-7 µs), which make them efficiently quenched by oxygen. Moreover, their photophysical features are very appropriate for designing dedicated analytical instrumentation based on solid-state electronics (blue LEDs, photodiode detectors) and inexpensive optical fibers (glass, plastic). It is not surprising therefore that such dyes are currently established as the paradigm for indicator-mediated optical sensing of molecular oxygen. To use luminescent dyes for fabrication of oxygen sensors, they must be immobilized in a proper solid matrix such as organic polymers (siloxanes, polystyrenes, poly(hydroxyethyl methacrylate)) or silica glasses and gels.3-12,15-19 These supports are frequently heterogeneous on a microscopic scale and give rise to (3) Wolfbeis, O. S.; Offenbacher, H.; Kroneis, H.; Marsoner, H. Mikrochim. Acta 1984, 1, 153. (4) Peterson, J. I.; Fitzgerald R. V.; Buckhold, D. K. Anal. Chem. 1984, 56, 62. (5) Optiz, N.; Graf H. J.; Lubbers, D. W. Sens. Actuators 1988, 13, 159. (6) Potyrailo, R. A.; Hieftje, G. M. Anal. Chim. Acta 1998, 370, 1. (7) Klimant I.; Wolfbeis, O. S. Anal. Chem. 1995, 67, 3160 and references therein. (8) (a) Demas, J. N.; DeGraff, B. A.; Xu, W. Anal. Chem. 1995, 67, 1377. (b) Carraway, E. R.; Demas J. N.; DeGraff, B. A. Langmuir 1991, 7, 2991. (9) Papkovsky, D. B.; Olah, J.; Troyanovsky, I. V.; Sadovsky, N. A.; Rumyantseva, V. D.; Mironov, A. F.; Yaropolov A. I.; Savitsky, A. P. Biosens. Bioelectron. 1991, 7, 199. (10) Liu, Y.-M.; Pereiro-Garcı´a, R.; Valencia-Gonza´lez, M. J.; Dı´az-Garcı´a, M. E.; Sanz-Medel, A. Anal. Chem. 1994, 66, 836. (11) Mills, A.; Thomas, M. D. Analyst 1998, 123, 1135. (12) Xu, W.; Kneas, K. A.; Demas, J. N.; DeGraff, B. A. Anal. Chem. 1996, 68, 2605. (13) Demas, J. N.; DeGraff, B. A. Anal. Chem. 1991, 63, 829A. (14) Orellana, G.; Quiroga, M. L.; de Dios, C. Trends Inorg. Chem. 1993, 3, 109. (15) Hartmann, P.; Leiner, J. P.; Lippitsch, M. E. Sens. Actuators B 1995, 29, 251. (16) Di Marco, G.; Lanza, M.; Campagna, S. Adv. Mater. 1995, 7, 468. (17) McEvoy, A. K.; McDonagh, C. M.; MacCraith, B. D. Analyst 1996, 121, 785. (18) McDonagh, C.; MacCraith, B. D.; McEvoy, A. K. Anal. Chem. 1998, 70, 45. 10.1021/ac980722x CCC: $15.00
© 1998 American Chemical Society Published on Web 11/07/1998
nonlinear Stern-Volmer plots for oxygen quenching as well as multiexponential emission decay curves.8,15 The immobilization procedures and the solid supports have significant effects on the performance of the optical sensors in terms of sensitivity and stability. Despite physical techniques such as dissolution,7,9,11,19 adsorption4,5,8,12,15 and entrapment in a porous network16-18 are very simple ways of immobilization and can give high yields at low costs; the durability of the sensing layers fabricated thereby is compromised since the luminophore can easily leach out the matrix. For instance, it has been found that the widely used oxygen indicator tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) is washed out its silicone support by organic solvents such as acetone.20 On the other hand, the electrostatic interaction10,21 between a charged dye and the oppositely charged functional group of the solid matrix results in considerable binding strength, but the indicator can also be displaced easily by the sample ions or solvent molecules. To increase the long-term stability of the sensitive phase, the luminescent indicator must be attached covalently to the surface of the solid support: no leaching (but some photobleaching) has been observed in those cases.3,22 However, most of matrixes do not have appropriate linkers for covalent bonding, so that the first step of such a dye immobilization usually involves modification of the surface in order to deploy reactive functional groups. Unfortunately, polymers with excellent oxygen permeability such as silicones do not usually lend themselves to surface modification, so that covalent attachment of indicators is extremely difficult.7 Celluloses, on the other hand, are easy to modify chemically but show low oxygen permeability.23 Heteropolycondensates of silicic acid such as silica gel or porous glass materials provide a robust nonswelling rigid support, the surface of which can be easily modified by a variety of chemical reactions.24 This feature makes the covalent immobilization of the dyes feasible, and the versatility and durability of the sensitive phase is increased. In this work, we report a comparative study on the spectralluminescent and oxygen-quenching properties in organic media of novel Ru(II) complexes covalently linked to amino-derivatized controlled pore glass (CPG) via sulfonamide bonds (Figure 1). The hydrophobicity of the indicator molecules has been varied thanks to the introduction of different chelating ligands to the basic bis[1,10-phenanthroline-4,7-bis(phenylsulfonate)]ruthenium(II) moiety. The resulting materials display strong emission above 600 nm which is effectively quenched by oxygen in both organic solvents and aqueous media. Different quenching models have been applied to account for the multiexponential luminescence decays and nonlinear Stern-Volmer plots observed. EXPERIMENTAL SECTION Chemicals. The synthesis (as sodium salts) and spectroscopic characterization of the oxygen indicators [Ru(s2d)3],4[Ru(s2d)2(acap)]2-, and [Ru(s2d)2(tdap)]2-, where s2d, acap, and tdap stand for 1,10-phenanthroline-4,7-bis(phenylsulfonate), 5-ac(19) Mills, A.; Lepre, A.; Theobald, B. R. C.; Slade, E.; Murrer, B. A. Anal. Chem. 1997, 69, 2842. (20) Bacon, J. R.; Demas, J. N. Anal. Chem. 1987, 59, 2780. (21) Li, P. Y. F.; Narayanaswamy, R. Analyst 1989, 114, 1191. (22) Hsu, L.; Heitzmann, H. U.S. Patent 4,712,865, 1987. (23) Stern, S. A.; Shah, V. M.; Hardy, B. J. J. Polym. Sci. B 1987, 25, 1263. (24) Colowick, S. P., Kaplan, N. O., Eds. Methods in Enzymology; Academic Press: New York, 1987; Vols. 135B, 136C, and 137D.
Figure 1. Chemical structure of the heterocyclic chelating ligands and the controlled pore glass-immobilized Ru(II) complexes.
etamide-1,10-phenanthroline, and 5-tetradecanamide-1,10-phenanthroline, respectively (Figure 1), have been previously described.25 CPG-Glycophase (460-Å pore diameter, 37-74-µm particle size) was from Pierce (Rockford, IL). Argon and oxygen of +99.995% purity were from cylinders supplied by L’Air Liquide (Madrid, Spain). All the organic solvents were of spectroscopic grade and used without further purification. Water was from a Millipore Milli-Q system. WARNING: most of the organic solvents used are flammable and/or toxic; therefore, a well-ventilated hood must be used in the preparation of mixtures and handling of all the solutions. Apparatus. Luminescence measurements were carried out with a Perkin-Elmer LS-5 spectrometer (U ¨ berlingen, Germany) equipped with a Hamamatsu R-928 red-sensitive photomultiplier (PMT) and controlled by a Perkin-Elmer 3600 data station. Luminescence decays were collected with an Edinburgh Instruments FL-900 time-correlated single-photon-counting spectrometer (Edinburgh, U.K.), using a 0.3-bar nitrogen-filled arc lamp and a Peltier-cooled (-29 °C) red-sensitive PMT (Hamamatsu R-955). Emission lifetimes were extracted from the luminescence decays using a nonlinear Marquardt fitting algorithm contained in the original software package from the manufacturer. The flow analysis system comprises a four-channel Gilson (Villiers-le-Bel, France) Minipuls 2 peristaltic pump equipped with Viton tubing (1.5-mm i.d). Teflon tubing was used between all components in the flow system. Two Aalborg (Monsey, NY) 150mm gas flow meters, individually calibrated by a volumetric method, were employed to measure the relative oxygen and argon flow rates (within (0.2%). All the experiments were carried out at atmospheric pressure (∼715 Torr) and 25 ( 2 °C temperature controlled by a PolyScience 9001 circulator (Niles, IL). Covalent Immobilization of Ru(II) Complexes in CPG. The oxygen indicator dyes were covalently bound to CPG beads previously functionalized with primary amino groups according to the following procedure: 1 g of CPG was stirred for 30 min in 25 mL of a 1% (v/v) solution of γ-(aminopropyl)triethoxysilane (Fluka, 96%) in acetone. After the solvent was removed at reduced pressure, the impregnated beads were dried at 115 °C for 12 h. Attachment of the Ru(II) complexes to the derivatized CPG was carried out by amide linkage between the sulfonate groups of the s2d ligands and the amino groups of the modified CPG.26 Since the sulfonate groups are considerably unreactive, they were (25) Garcı´a-Fresnadillo, D. Ph.D. Thesis, Universidad Complutense de Madrid, Madrid, 1996; pp 184-196, 325-366.
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Figure 2. Optosensing manifold for oxygen monitoring in organic solvents using porous glass beads with covalently bound luminescent Ru(II) complexes.
previously activated to the form of sulfonyl bromide, which was allowed to react in situ with silver trifluoromethanesulfonate to yield the mixed anhydride, a highly reactive species toward Nand O-nucleophiles. A typical immobilization procedure is given herein: 0.01 mmol of Na2[Ru(s2d)2L] (L ) heterocyclic chelating ligand, Figure 1) was mixed with 0.16 mmol of PBr5 (Aldrich, 95%) under an argon atmosphere. After 15 min of stirring, 30 mL of anhydrous dimethylformamide (DMF) was added dropwise. The resulting red solution was refluxed for 1 h and then temperature was slowly decreased to 0 °C. In this moment, 0.18 mmol of silver trifluoromethanesulfonate (Fluka, + 98%) was dissolved in 10 mL of anhydrous DMF, added dropwise to the red solution, and the mixture was stirred for additional 30 min. The sulfonic anhydride solution was taken with a syringe under dry argon and was filtered (Millipore, Millex-FGS PTFE membrane filter, 0.2-µm pore size, 29-mm diameter) into a round-bottom flask containing a slurry of 0.25 g of amino-CPG and 18 mmol of anhydrous pyridine in 10 mL of DMF at 0 °C under argon. The mixture was stirred for 12 h at room temperature. The covalently dyed CPG beads were filtered, washed successively with DMF, water, methyl alcohol, and acetone, and dried at reduced pressure (0.005 Torr) overnight. No attempts were made to quantify the small amount of indicator bound to the CPG surface, but some relative indication is given in the Results and Discussion section. Measuring Procedure. The indicator-containing CPG beads were packed in a 100-µL Hellma (Mu¨llheim, Germany) flowthrough cell (model 176.052-QS). A small piece of nylon netting was placed at the bottom of the cell to prevent displacement of the particles by the carrier solvent. The fluorescence flow cell was then connected to the flow system; the glass beads were loaded with the aid of the peristaltic pump and allowed to settle to be sure that the particles were in the light path. The complete measuring setup is shown in Figure 2. The peristaltic pump was used to generate a stream of the organic solvent with different concentrations of dissolved oxygen passing continuously from and to the gas saturation chamber through the optical sensor. An oxygen-free carrier was pumped initially in order to obtain a stable reference emission (I0). The concentration of dissolved oxygen in each solvent was calculated using the Ostwald or Bunsen oxygen solubility coefficients27 and assuming (26) Orellana, G.; Garcı´a-Fresnadillo, D.; Moreno-Bondi, M. C.; Xavier, M. P. Span. Patent Appl., 9602181, 1996. (27) Battino, R.; Clever, H. L.; Young, C. L. IUPAC Solubility Data Series; Pergamon Press: Oxford, 1981; Vol. 7.
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Figure 3. Luminescence spectra in deoxigenated methanol of covalently immobilized [Ru(s2d)3]4- (s), [Ru(s2d)2(acap)]2- (- -), and [Ru(s2d)2(tdap)]2- (- - -); λexc ) 475 nm.
Henry’s law obedience; to that end, atmospheric pressure readings were corrected for the vapor pressure of each solvent at 25 °C.28 RESULTS AND DISCUSSION Spectral Features of the Immobilized Indicators. The luminescence spectra of the three Ru(II) complexes covalently immobilized on CPG are depicted in Figure 3. When excited at 475 nm, all of them show a maximum around 610 nm in methanol as well as in the other selected organic solvents. A weak band at ∼530 nm, attributable to excitation light scattered by the solid support, is evident in two of the spectra. The intensity of the scattering depends on the settlement of the dyed glass beads inside the spectrofluorometric flow cell, so that no background correction of the emission spectra has been attempted. Nevertheless, it was checked that its contribution at the analytical wavelengths (>615 nm) is negligible. The emission band arises from deactivation of a triplet metalto-ligand charge-transfer (3MLCT) excited state, although the heavy atom blurs somewhat its spin label.13,14 Table 1 summarizes the luminescence maximums of the three complexes, showing no significant variations with changes in solvent polarity. Notwithstanding with the polarity of the MLCT excited state, the local environment provided by the glass surface seems to shield it from the expected solvent effect. However, since the emission quantum yields of the three complexes are similar (0.17 ( 0.03 in deoxygenated water), and their absorption coefficient are similar too (∼29 000 L mol-1 cm-1 in water), the observed luminescence intensity of the CPG-bound complexes must be directly related to the amount of dye covalently attached to the glass surface. The relative emission intensities of the dyed beads (e.g., Figure 3) are 1:8:12 in any of the organic solvents selected, for [Ru(s2d)2(tdap)]2-, [Ru(s2d)3]4-, and [Ru(s2d)2(acap)]2-, respectively. Therefore, such relationship would correspond to the different loading of the support. The efficiency of the sulfonamide linkage formation might be related to the steric volume of the different metal complexes. A comparative study of the spectral features of the luminescent indicators covalently immobilized on CPG and in solution is (28) Hodgman, C. D. CRC Handbook of Chemistry and Physics, 42nd ed.; The Chemical Rubber Publishing Co.: Cleveland, 1961.
Table 1. Luminescence Wavelength Maximums (in nm) of the CPG-Immobilized Ru(II) Complexes in Organic Solvents (λexc ) 475 nm) dye
methanol
chloroform
cyclohexane
toluene
n-hexane
[Ru(s2d)3]4[Ru(s2d)2(acap)]2[Ru(s2d)2(tdap)]2-
620 616 605
612 605 605
622 616 601
620 615 601
622 615 601
Figure 4. Dissolved oxygen dose-luminescence plots for [Ru(s2d)3]4- (a), [Ru(s2d)2(acap)]2- (b), and [Ru(s2d)2(tdap)]2- (c) immobilized onto CPG, in methanol (9), chloroform (2), cyclohexane (1), toluene ([), and n-hexane (f). The solid lines represent the best fit of the experimental points to the two-site quenching model (eq 1; see text).
prevented by the lack of solubility of the anionic complex salts in chloroform, cyclohexane, toluene, and n-hexane. The emission maximums in methanol for [Ru(s2d)3],4- [Ru(s2d)2(acap)]2-, and [Ru(s2d)2(tdap)]2- are 616, 615, and 614 nm, respectively, values similar to those measured for the CPG-bound dyes (Table 1). In a solid matrix, the environment around the luminescent complexes cannot reorientate as fast as in fluid solution, so that a less stabilized excited state is produced, with concomitant blue shift of the emission (rigidochromism).14 However, the polar surface of CPG seems to be able to solvate efficiently the charge-separated 3MLCT excited state and rigidochromism is not observed in this case. Performance of the O2-Sensing Phases in Organic Solvents. The relative luminescence intensity of the immobilized dyes as a function of the oxygen concentration in the different organic solvents is depicted in Figure 4. The three sensing heads display a higher oxygen sensitivity in methanol than in any of the hydrocarbon solvents tested (chloroform, cyclohexane, toluene, n-hexane). Moreover, quenching by oxygen is higher for the luminescent Ru(II) complex containing an acap ligand than for the homoleptic s2d complex in all the above-mentioned solvents, with the metal complex containing a tdap ligand the least sensitive of the family. Since the emission lifetimes of the three dyes are equal (0.31, 0.32, and 0.30 ( 0.01 µs in aerated methanol at 25 °C, for [Ru(s2d)3],4- [Ru(s2d)2(acap)]2-, and [Ru(s2d)2(tdap)]2-, respectively), the different oxygen-quenching plots for the CPG-bound complexes investigated must be directly related to the dye loading, if we assume that the quenching rate constants are similar for the three indicators, as is the case for the other Ru(II) complexes.29 This fact provides new evidence that the oxygen-quenching process occurs indeed by diffusion of the
reaction partners on the supporting surface30 and not by collision with the homogeneously dissolved oxygen: for a given concentration in a solvent, a certain amount of molecular oxygen will be adsorbed onto the CPG and will be able to deactivate the photoexcited Ru(II) complex provided they can collide within its lifetime. Calibration plots for the three luminescent dyes in CPG are not linear in the whole concentration range up to oxygen saturation (Figure 4). Curved Stern-Volmer quenching plots are usually observed with supported indicators and this is related to heterogeneity at the molecular level.8 Analysis of the experimental data must begin by establishing a proper quenching model. We have evaluated two mechanistic models previously described by Carraway et al.,8b namely, (i) multisite populations of immobilized complexes with its corresponding quenching constants and (ii) quenching determined by oxygen adsorption onto the solid support following a Freundlich isotherm. Excellent fits were always obtained using the first one, i.e., assuming the existence of two independent binding sites both being quenched by oxygen diffusion but with different rate constants (eq 1), where f0i is the
I0/I )
(
f01
1 + KSV1[Q]
+
f02 1 + KSV2[Q]
)
-1
(1)
fraction of the total emission from each population under unquenched conditions and KSVi are the associated Stern-Volmer (29) Garcı´a-Fresnadillo, D.; Georgiadou, Y.; Orellana, G.; Braun, A. M.; Oliveros, E. Helv. Chim. Acta 1996, 79, 1222. (30) Mingoarranz, F. J.; Moreno-Bondi, M. C.; Garcı´a-Fresnadillo, D.; de Dios, C.; Orellana, G. Mikrochim. Acta 1995, 121, 107.
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Table 2. Parameters of the Best Fit of the Experimental Data for Deactivation by Molecular Oxygen of CPG-Immobilized Ru(II) Indicators (Figure 4), to a Two-Site Quenching Model (Eq 1; See Text) dye [Ru(s2d)3
]4-
[Ru(s2d)2(acap)]2-
[Ru(s2d)2(tdap)]2-
a
solvent
KSV1 (mM-1)
methanol chloroform cyclohexane toluene n-hexane methanol chloroform cyclohexane toluene n-hexane methanol chloroform cyclohexane toluene n-hexane
0.26 0.14 0.10 0.11 0.07 0.24 0.17 0.12 0.11 0.09 0.020 0.014 0.012 0.006 0.009
f01a
0.26
0.24
0.85
KSV2 (mM-1) 2.24 1.40 2.26 1.30 2.07 3.21 1.84 2.48 1.80 2.22 0.93 1.19 1.68 1.00 1.24
f02a
r
0.999 0.999 0.74 0.999 0.999 0.999 0.999 0.999 0.76 0.999 0.999 0.999 0.997 0.999 0.15 0.999 0.999 0.999
Estimated error (0.05.
parameters R and n are proportional to KSV and the intensity of adsorption, respectively) also produced excellent fits for the experimental data of the three complexes (r > 0.997). The calculated curves cannot be distinguished from those shown in Figure 4. However, the lack of an experimental basis for the Freundlich isotherm in the system under study, or other indicator dyes adsorbed onto silica, zeolites, or porous Vycor glass, makes this nonlinear model less attractive than the multisite one. In addition, we have also evaluated the relative contributions of static and dynamic quenching for the oxygen-sensing phase containing covalently bound [Ru(s2d)2(acap)]2-. To that end, we measured the luminescence decay profiles of the dyed CPG beads as a function of the oxygen concentration in methanol (Figure 5). The kinetic profiles turned out to be complex at all the analyte levels: three-exponential functions were required to produce a satisfactory fitting of the experimental data. To compare the Stern-Volmer lifetime and intensity plots and assess the static quenching contribution, we have reduced the multicomponent decays to a single quantity, i.e., the preexponential weighted mean lifetime defined according to eq 2,31 where Bi and τi are the
τM )
Figure 5. Normalized luminescence intensity and preexponential weighted emission lifetime of CPG-immobilized [Ru(s2d)2(acap)]2as a function of the oxygen concentration in methanol at 25 °C and a total presure of 715 Torr.
quenching constants for each component. The best fitting parameters for the three immobilized complexes are summarized in Table 2. There is currently a general agreement8 in that such a model actually represents an oversimplification of a much more complex situation, which would comprise a distribution of indicator molecules on the surface of the glass beads. A bimodal distribution of ruthenium(II) complexes (eq 1) produces excellent fits without having recourse to subsequent refinements of the quenching model. The validity of the model is further confirmed by the singularity of the f0i values obtained for each sensing phase (Table 2). Regardless of the organic solvent that baths each type of dyed glass beads, the relative populations of the two indicator sites quenched by oxygen remain unchanged, being 3 times higher the contribution of the most heavily quenched one. CPG-bound [Ru(s2d)2(tdap)]2- is exceptional in that the major component of its luminescence is formed by these sites weakly deactivated by the analyte. The quenching model based on the empirical Freundlich gas adsorption isotherm (I0/I ) 1 + R[O2]1/n, where the fitting 5188 Analytical Chemistry, Vol. 70, No. 24, December 15, 1998
∑B τ /∑B i i
i
(2)
preexponential factors and the emission lifetimes, respectively, of the different components extracted from the nonlinear leastsquares fit. The coincidence of the relative intensity and lifetime plots shown in Figure 5 demonstrates that quenching of [Ru(s2d)2(acap)]2- attached to the CPG surface by molecular oxygen is purely dynamic. This finding agrees with the low degree of surface coverage achieved by covalent binding of the ruthenium dye. Since the best response was obtained in every case with the immobilized [Ru(s2d)2(acap)]2- complex, a thorough analytical caracterization of the sensing head containing the indicator was further performed. Analytical Features of the O2 Sensor Containing [Ru(s2d)2(acap)]2-/CPG. A typical oxygen dose-response plot in methanol for the luminescent sensor fabricated with CPG beads and covalently attached [Ru(s2d)2(acap)]2- is depicted in Figure 6. The dynamic range of the sensor is 9.2 × 10-3-8.0 mM oxygen in methanol (0.12-100% of the maximum solubility), but the highest sensitivities are obtained between 9.2 × 10-3 and 8.3 × 10-4 mM (0.12-9%) concentration. Different experiments performed by measuring the emission signal with increasing/ decreasing values of the oxygen concentration showed no hysteresis effects (Figure 6, inset). Similar response functions were obtained in the other organic solvents tested; the analytical figures of merit of the optosensor are collected in Table 3. The precision of the oxygen sensor was evaluated at oxygen concentrations of 9, 55, and 100% of the saturation level in each organic solvent. Relative standard deviations (RSD, n ) 6) are summarized in Table 3. The detection limits were calculated as the concentration of oxygen that produces an analytical signal equal to the blank one plus 3s, where s is the standard deviation of 10 I0/I ratios in the absence of analyte. The response time of the sensing head depends on the gas mixing process, the time (31) Carraway, E. R.; Demas, J. N.; DeGraff, B. A. Anal. Chem. 1991, 63, 332.
for a concentration increase step was ∼2 min in all the organic solvents tested. Finally, over more than four months of repeated use, the immobilized complexes did not leach out of the CPG using either water or organic solvents, as is to be expected from a covalent bond between the luminescent indicator and its support. Moreover, the sensing particles proved to be photochemically stable, both in the presence and in the absence of oxygen, under continuous irradiation for more than 10 h with the spectrofluorometer xenon lamp (2.7 × 1014 photons s-1 at 475 ( 2.5 nm, as determined by actinometry with Aberchrome-54032). CONCLUSIONS Figure 6. Oxygen response function of CPG-[Ru(s2d)2(acap)]2- in methanol. Inset: forward and reverse step changes in the oxygen concentration; λexc ) 475 nm, λem ) 616 nm. Table 3. Analytical Characteristics for Oxygen Sensing in Organic Media with [Ru(s2d)2(acap)]2Covalently Bound to CPG solvent
RSDa (%)
[O2] dynamic rangeb (M)
DLc (µM)
t90d (min)
methanol chloroform cyclohexane toluene n-hexane
2.0 4.0 6.0 3.0 3.0
3.0 × 10-5-7.8 × 10-3 1.3 × 10-4-7.8 × 10-3 1.2 × 10-4-9.4 × 10-3 6.8 × 10-5-8.7 × 10-3 1.7 × 10-4-1.3 × 10-2
6.2 27 20 12 27
2.4 2.9 1.7 1.7 1.2
a Relative standard deviation (n ) 6). b Expressed as quantification limit ([O2] that produces an analytical signal equal to the blank signal plus 10s, where s is the standard deviation of 10 I0/I ratios in absence of analyte) to oxygen saturation in the solvent. c Detection limit. d Response time for a 90% signal change.
required to saturate the carrier solvent, and the oxygen diffusion rate through the CPG beads (see Figure 2). With our experimental setup, the measured t90 (time to reach 90% of its final value) (32) Kuhn, H. J.; Braslavsky, S. E.; Schmidt, R. Pure Appl. Chem. 1989, 61, 187.
Covalent binding of sulfonated ruthenium(II) complexes to amino-derivatized porous glass allows direct luminescent monitoring of molecular oxygen in any solvent, without leaching or chemical attack to indicator polymer layers. The resulting calibration curves are heavily nonlinear due to the microheterogeneous environment around the photoexcited indicator and not to a contribution of static quenching from adsorbed oxygen molecules. Detection limits as low as 6.2 µM oxygen have been obtained with a conventional spectrofluorometer; lower limits might be achieved using dedicated instrumentation or blue laser excitation. Further work is currently in progress in order to apply the luminescent sensor to optical biosensing based on oxygen transduction in nonaqueous media. ACKNOWLEDGMENT This project has been funded by the Spanish government agency CICYT under Contract AMB95-0689-C02. M.P.X. thanks the Spanish Ministry of Education and Culture for a FPI doctoral grant.
Received for review July 6, 1998. Accepted September 25, 1998. AC980722X
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