Anal. Chem. 1991, 63,797-802
707
Determination of the Transduction Mechanism for Optical Sensors Based on Rhodamine 6G Impregnated Perfluorosulfonate Films Using Steady-State and Frequency-Domain Fluorescence Kevin S. Litwiler, Pamela M. Kluczynski, and Frank V. Bright* Department of Chemistry, State University of New York at Buffalo, Buffalo, New York 14214 Multifrequency phasemodulation fluorescence spectroscopy is used to investigate the dynamics of thln Nafion films impregnated with rhodamine 6 6 (R6G). Using this approach, we explore the photophysics of these films as a function of R6G concentration, water content, Cu( I I ) quencher concentration, and film preparatlon procedure. From this, we infer how these films function as chemical recognltion elements in fiber-optic-based sensors. The decay kinetics for ail films studied are best described by a slmple Moexponential decay law, a result of ground-state dimer formation.
INTRODUCTION In the areas of biocompatibility, catalysis, and chromatography, there is significant interest in improving our understanding of how surface-associated species function in comparison to their bulk solution counterparts. However, probing surfaces can present unexpected difficulties and shortcomings. Consequently, many analytical techniques (e.g., scanning electron microscopy, X-ray photoelectron spectroscopy, fast atom bombardment mass spectrometry, secondary-ion mass spectrometry, total internal reflectance IR spectroscopy, contact angle, chemical activity, and differential scanning calorimetry) are now used to study interfaces. Ideally, the chosen technique should be sensitive, selective, information-rich, versatile, nondestructive, and capable of examining the surface in its native environment. Fluorescence spectroscopy has a well-established (1)track record for investigating solution-phase systems, and because fluorescence is inherently information rich (2,3), it has been used also to study surface processes. For example, structural and morphological changes a t an interface are often detected and quantified by shifts in fluorescence excitation/emission spectra. However, additional, more detailed information can be recovered by observing changes in the kinetics of fluorescence processes. For this reason, time-domain fluorescence has been used to study such things as the rates of energy transfer and electron transfer to and from a surface and a local fluorescent center (4-6). Unfortunately, acquisition of high-quality time-domain decay data can require unreasonably long periods of time (>1 h). Over this relatively long time scale, changes in the interfacial species can occur and, because acquisition is too slow, would be missed or “smeared out”. One solution to this problem is to perform the analysis in the frequency domain (7-10). By using state-of-the-art parallel processing instrumentation, complete fluorescence decay profiles can be acquired in less than 1s (11). However, because of the typical method of modulating the excitation beam (i.e., a Pockel’s cell), one must make modifications to frequency-domain instrumentation to allow the analysis of surfaces without biasing the experimental data. *Author to whom all correspondence should be sent. 0003-2700/91/0363-0797$02.50/0
For the past 3 years, our group has been developing optical sensors that use fluorescence as the information carrier (12-16). During this same period, we have become concerned with determining how (on a fundamental level) analyte molecules interact with fiber-optic sensing elements to yield an analytically useful signal. In an effort to improve our understanding of how fiber-optic chemical sensors work, we have focused on determining the details of the photoemission processes occurring at the sensing element. Our contention is that once the response mechanism of a sensor (and causes of activation or inhibition) is understood, a more stable device, which is most selective and sensitive, can be constructed based on predictive theory rather than trial and error. As an example system, we decided to investigate a previously described optical sensor (17, 18) based on thin Nafion films impregnated with rhodamine 6G (R6G). Hieftje and co-workers reported earlier that this simple scheme could be used for quantification of humidity (17) and metal ions (18). In both cases, changes in steady-state fluorescence (from the R6G) were used to determine the humidity or ion concentration. However, spectral information alone provides little chemical insight ink! the details of the actual transduction process. Time-resolved experiments were also performed in the earlier work (13,but the resolution of the equipment did not allow complete interpretation of the results. In this paper, the necessary modifications and the use of multifrequency phase and modulation fluorescence for surface studies are described and discussed. Specifically, we report on multifrequency phase and modulation experiments aimed a t determining the details of signal transduction in these R6G-impregnated Nafion film-based sensors.
THEORY The theory of frequency-domain fluorescence spectroscopy has been described in detail elsewhere (7-10). Additional details on steady-state fluorescence, dimer formation, and static and dynamic quenching can be found in ref 1. For completeness, we review briefly the salient features of multifrequency phase and modulation fluorescence. Following excitation with an optically short pulse of light, the time-dependent emission intensity, I ( t ) , is given by a multiexponential decay of the form
where ai is a preexponential factor that represents the fractional contribution (mole fraction) of component i , with lifetime T ~ .The fractional contribution to the total fluorescence intensity of each species is
vi)
In the frequency domain, the sample under study is excited with sinusoidally amplitude-modulated light (7-10). The 0 1991 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 8, APRIL 15, 1991
resulting fluorescence is equal in frequency to the excitation waveform but phase-shifted (0) and amplitude-demodulated (M)to an extent dependent on the fluorescence lifetime(s) of the sample (7-10). For any decay process, the calculated (subscript c) values of O and M a t any angular modulation frequency (w = 2nf, f is the linear modulation frequency) are given by (7-10) 8,(w)
= tan-’
(g)
(3)
(4) where S ( w ) and C ( w ) are given by the sine and cosine Fourier transforms (7-10)
By using recently developed instrumentation ( 1 1 ) based on the parallel acquisition of data, sequential measurement of Om(w) and Mm(o)at several modulation frequencies is no longer necessary. Rather O,(w) and Mm(w)are calculated from the harmonic content of the fluorescence response of the sample after excitation with a pulsed source using Fourier transform algorithms ( 1 1 ) . In this approach, the sum of the squared differences between the calculated Oc(o)and M,(w) values and measured OJw) and M,(w) values are minimized by nonlinear least-squares analysis (7-10) using the x2 function:
(7) Here ugand vM are the variances in phase angle and demodulation, respectively, and D is the number of degrees of freedom. When uo and V M accurately reflect the uncertainty in the experimental 19 and M measurements, the goodness of fit is judged by the (1)closeness of x2 to unity and (2) random distribution of the residual phase angle and demodulation factor about zero.
EXPERIMENTAL SECTION Nafiion 117 commercial films (0.007-in.thick, Lot No. 00321lT), Nafion solution (5% w/w in alcohols), and rhodamine 6G (-99% pure) were purchased from Aldrich (Milwaukee, WI). Eosin isothiocyanate was purchased from Molecular Probes (Eugene, OR). Aminopropyl-controlled pore glass (particle mesh size 200/400,mean pore diameter 182 A i 4.7%, Lot No. 03C06 AMP) was from Controlled Pore Glass, Inc. (Fairfield, NJ). Copper sulfate was purchased from J. T. Baker, Inc. (Phillipsburg, NJ). All reagents were used as received. Stock solutions were prepared in distilled, deionized water. Eosin isothiocyanate (EITC) covalently attached to aminopropyl-derivatized controlled pore glass (CPG) was prepared by gently stirring 0.1 g of CPG in 200 nM EITC for 10 min. EITC-CPG was then washed with copious amounts of water, creating beads that were light pink with approximately 13 nmol of EITC/g of CPG (6.9 X molecules/nm*). Nafion films for study were prepared by two different protocols. First, for cast films, 50 pL of Nafion solution was pipetted directly onto a chromic acid cleaned quartz slide (1cm X 1 cm) and the solvent allowed to evaporate slowly. The films were then washed in dilute nitric acid and water. For commercially prepared films, we followed a boiling nitric acid protocol described elsewhere (19). Rhodamine 6G impregnated Nafion (R6G-Nafion) films were prepared by immersing (for 10 min) Nafiion films in 20 mL of fresh aqueous R6G solutions. The films were then removed, washed with water, and stored in water overnight. For our studies, we investigated both wet and dry films. “Wet“ films were analyzed directly after removal from storage in water.
“Dry”films were prepared by placing wet films in a 60 “C oven for 15 min. Qualitatively, dry fiis appear yellow, while wet films appear red. Absorbance measurements were made by using a Milton Roy Model 1201 UV/vis spectrophotometer. Steady-state and frequency-domain fluorescence experiments were carried out by using an SLM 48000 MHF equipped with a modified sample chamber (20). The sample chamber was modified by replacing the cuvette holder with a bifurcated quartz optical fiber (General Fiber Optics) that is positioned by using an r,y , z translation stage (Newport, LP-05). The modulated excitation beam was focused into one arm of the fiber (600-pm core diameter). It passed through and exited the fiber optic to strike the sample or reference. Fluorescence was collected by a second fiber (600 pm core diameter), delivered to focusing optics through an emission filter (Oriel; long pass or band-pass), and finally imaged onto the photocathode of a photomultiplier tube detector. A Coherent (Innova 90-6) argon-ion laser was used as the excitation source (496.5-nm line). The multifrequency phase and modulation t r a m were acquired as described elsewhere (11). For all experiments, the Pockel’s cell was operated at a repetition rate of 5 MHz. Typically, data were acquired for 1 min.
RESULTS AND DISCUSSION Characterization of the Instrument. Accurate fluorescence lifetime work requires the use of well-characterized reference lifetime standards (21). For conventional liquid-phase studies, the common practice is to use dilute solutions of fluorophores (with similar spectral contours to the system under study) that decay with a single lifetime (21). T o use these existing reference standards with confidence, while measuring the fluorescence decay kinetics at surfaces, we must first verify that no biases are induced when comparing morphologically different samples and references (i.e., solutions to surfaces). In a previous communication (22),we described some of the instrumental artifacts unique to studies of surface-immobilized species that one can observe for frequency-domain fluorescence. Most of these artifads were traced to the spatial inhomogeneities within the modulated beam profile (22). We found that short lengths of multimode optical fiber alleviated most of these problems. However, at that time, we had not developed a “surface system” that was clearly described by a single decay time. Therefore, we could not determine absolutely if there were any additional biases (22). Because the samples in this study, Nafion films, are morphologically different from solutions, our initial challenge was to verify that by using the optical fiber (22)there are no biases due to such differences. However, in order to meet this challenge, we needed a surface-based model system with “known”single-exponential decay kinetics. If we were to have such a system, we could determine immediately (21) if our scheme (22) would allow a liquid-phase reference lifetime standard to be used to recover accurately the dynamics of surface-bound species. Unfortunately, literature reports of surface-immobilized species having simple, single-exponential, and reproducible decay kinetics, to our knowledge, do not exist. After some trial and error, we found one surface system that is (1) described by a single-exponential decay law, (2) inexpensive, (3) intensely fluorescent, (4)excited with visible light, ( 5 ) stable, and (6) easily prepared. Using aqueous R6G solutions as the reference ( T = 3.960 ns), we established that immobilized EITC on CPG was described by a single-exponential decay law ( T = 2.438 f 0.002 ns). Thus, because single-exponential decay kinetics were recovered from this system, we concluded that dilute fluorophore solutions could be used with confidence in all of the following experiments as the reference lifetime standard (21). Application toward Nafion-Based Sensors. To improve our understanding of how analyte interaction with R6G-impregnated Nafion films leads to the analytical signal, we in-
ANALYTICAL CHEMISTRY, VOL. 63,NO. 8, APRIL 15, 1991
799
-..-Commercial Film -Solution Cast Film 1.51
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0.5
0.0 2w
100
,-. x 80
Wavelength (nm)
-
v
Figure 1. Absorbance spectra of rhodamine 6G in commercial (- -) and solutiorrcast (-) Nafion films. Panel A shows spectra of wet films. Panel B shows spectra of dry films. The concentration of R6G in the loading solution was 10 pM.
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(7,) versus the concentration of R6G used to prepare the R6GNafbn film results are shown. film. Wet (0)and dry (0)
:
B
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Concentration o f R 6 G Costing Solution (mM)
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vestigated the time-resolved fluorescence of this system. Specifically, we investigated the fluorescence decay kinetics as a function of R6G, Cu(I1) concentration, and water content. Further, we carried out a comparison study between solution-cast and commercial Nafion films. Results for solution-cast films are discussed in the bulk of the remaining text; a brief discussion of the commercial preparations is included at the end. In all of the following experiments, the term "dry" refers to film preparations that have been kept in the oven. "Wet" films have been soaked in water prior to measurement. No attempt was made to ramp the humidity or quantitate the amount of water or Cu(I1) actually in the films. Effects of Dye Concentration and Water Content. With respect to fiber-optic-based sensing, the first use of R6G-Nafion films was as the chemical recognition element for a humidity sensor (17). To understand, in detail, how this sensor functioned, we investigated a wide range of R6G concentrations on wet and dry films. Steady-state absorbance and fluorescence spectra were studied first. The absorbance maxima and peak shape for wet films (Figure 1,panel A) (518 nm with a shoulder at 485 nm) are very similar to solutions of R6G in any liquid (polar and nonpolar, acidic and basic) that we examined. In contrast, the absorbance spectra of dry films, regardless of R6G concentration, are dramatically blue-shifted, have a different shape, and have significantly
lower absorptivity (Figure 1, panel B). Similarly (Figure 2), we observe clear changes in the emission contours of the wet and dry films. Because the absorbance/emission characteristics of R6G solutions are very similar in any solvent, we conclude that major changes are occurring in the local R6G environment in these films as water is removed. Beyond this, however, few concrete conclusions can be drawn from the absorbance and steady-state emission spectra alone, but it appears that there are at least two forms of R6G and each form exists in the ground state. In an effort to gain further insight into the system, we turned to frequency-domain fluorescence spectroscopy (7-10). Figure 3 shows a typical multifrequency phase and modulation data set (solid points) and corresponding fits to single-exponential (-) and double-exponential (- - -) decay models. Clearly, the double-exponential model yields the best fit. We also investigated continuous unimodal lifetime distributions (23),but they too failed (results not shown) to yield a fit superior to the double-exponential model. Moreover, we found that, regardless of the concentration of R6G or the water content in the film, the emission decay kinetics were always best fit by a double-exponential model. These results are consistent with the existence of only two unique R6G species (emissive centers). That is, either the R6G is interacting with itself or with the Nafion polymer in a significant manner, creating not a distribution of species but two discrete "forms" of R6G. Figure 4 shows the recovered fractional contribution to the total fluorescence intensity of the shorter lived component (rl)as a function of R6G concentration for wet and dry films. Similarly, Figure 5 reports the recovered lifetimes of both components as a function of R6G concentration and water content. Comparing four identically prepared films, the relative standard deviations in f i , rl, and r2 were 4.2%, 6.5%, and 2.5%, respectively. In general (Figure 4), fi increases in a sigmoidal fashion as R6G is added to the film. Also, on average, f l is about 11% higher for the wet films. If these values are expressed in terms of fractional concentrations (ai)
vi)
800
ANALYTICAL CHEMISTRY, VOL. 63, NO. 8, APRIL 15, 1991 0-071
0-072 0-071 W-WT~
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instead of fractional intensities (plots not shown), they also increase sigmoidally, from 8% to 80% for the dry films and 9% to 90% for the wet films. Thus, a t low concentrations of R6G, there is only a small contribution from the short-lived component (I& However, as R6G is added, more species corresponding to this short-lived component are created. A t very high R6G concentrations, the fractional contribution of the short-lived component (fJ reaches a limiting value (= 50-60%) and is clearly formed more in the wet films (Figure
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In general, all fluorescence lifetimes decrease as R6G is increased (Figure 5). This is most likely a result of some form of concentration quenching between the R6G molecules and is not totally unexpected. Interestingly, a t low concentrations of R6G (Figure 5), for either the wet or the dry films, we find that the longer lived decay time approaches that for R6G in water (I = 3.96 ns) (22). The shorter lived component, in contrast, approaches a value near 2 ns, but its contribution to the total emission becomes vanishingly small as R6G is decreased. On the basis of these experimental results, the response of the previously reported humidity sensor (I7) is apparently caused by major changes in the absorbance spectra (Figures 1 and 2) and more moderate changes in the lifetimes and relative contributions of the two emitting species with the addition of water. However, the question now becomes, what are the two emitting species? Previously, Hieftje and co-workers proposed (17) that there were solvated and unsolvated forms of R6G within the Nafion films. However, as we have shown here, both dye concentration and degree of hydration affect the relative speciation. As an alternative proposal, we suggest a new mechanism. It has been reported that based on electrochemical and absorbance measurements methylene blue forms a dimer in Nafion films at high concentrations (24). We propose a similar mechanism to operate in our system. Here 72 and T~ correspond to emission of the monomer and dimer species, respectively. In the ground state, we propose R6G forms a dimer with a nearby R6G molecule. The probability of this formation is, of course, dependent on the concentration of the R6G in the film. As the amount of R6G increases, the concentration of the dimer increases also (Figure 4). After the monomer and the dimer absorb a photon, becoming excited into the singlet manifold, both can emit a photon and be detected as fluorescence from their respective states. This, we propose, is the source of the red-shifted shoulder in the emission spectra a t high R6G concentrations (Figure 2). Also, because we found no evidence of excited-state reactions, the dimer must be stable during its excited-state lifetime. Since at low levels of R6G the lifetime of the lifetime of the longer lived component approaches a lifetime near that of R6G in water, we propose that this component must correspond to the R6G monomer. Thus, the shorter lived component (TJ
1
WET
Figure 6. Proposed model for wet and dry R6G-Nafion films.
would correspond to the dimer. The dimer has an intrinsically shorter lifetime compared to the monomer because there are more modes of deactivation and therefore the dimer has a faster competing rate against emission. Assigning the shorter lived component to the dimer also agrees with the increase in the relative concentration of the first component as the concentration of R6G is increased (Figure 4). The alternative to this model is that R6G is associating with Nafion (the sulfonate groups) to form a second "species" with its own characteristic photophysics (lifetime). However, with this model, the concentration of unaffected R6G (72) would increase with increasing R6G concentration in the film. In other words, the first amounts of R6G added to the film should associate with the Nafion where its lifetime would be shortened. Subsequently, added R6G would be "free" R6G. This is not the case according to the experimental data (Figure 4). Clearly, water affects both the fractional concentrations and the lifetimes of the components (Figures 4 and 5). One can see (Figure 4) that by increasing the water concentration that one simultaneously increases the amount of dimer formed. Therefore, in some way water must stabilize or facilitate dimer formation. This could be explained by the following scenario (Figure 6). When water is removed from the film, the R6G molecules come in closer contact with the anionic sulfonate groups at the membrane cavity walls. At the walls, ion-pairing interactions dominate the intermolecular forces (R6G is a cation), and the monomer is formed preferentially (one cation to one anion). In contrast, when water is present in the Ndion cavities, the ion pairing is disrupted somewhat due to the more stable hydrated R6G. The hydrated monomer may still associate with the wall, but the attraction would be coulombic rather than from the very strong ionic forces. Once the R6G is no longer ion paired, it is free to form a dimer, which according to our data (Figure 4) is slightly more stable than the hydrated monomer. Once formed, the dimer may diffuse farther into the water pool of the Nafion cavity since it is doubly charged and is repelled from the net positive charge near the wall. Decay Kinetics versus Quencher Concentration. With
ANALYTICAL CHEMISTRY, VOL. 63, NO. 8, APRIL 15, 1991
Table I. Recovered Parameters from Stern-Volmer Plots of Cu(I1) Quenching of Wet and Dry RGGNafion Films"** species
R2C
T~," ns
monomer, dry dimer, dry monomer, wet dimer, wet
0.9974 0.9939 0.9994 0.9976
3.45 0.90 3.72 1.50
k,: M-'s-l 2x 12 x 3x 44 x
10'0
1010 1010 10'0
Film prepared from 10 pM R6G. bCu(II) concentration varied from 0 to 10 mM. 'Correlation coefficient for Stern-Volmer plot. Lifetime without quencher. e Apparent bimolecular quenching constant (f1570).
respect to fiber-optic sensing, the second use of the R6GNafion films was as a metal-ion sensor (18). As an example, Cu(I1) gave a nearly linear decrease in fluorescence over the range from 1.2 r M to 12 mM. At this time, it was suggested that the response was due to quenching of the R6G. However, now knowing that there are two species in the film (i.e., the monomer and the dimer), dynamic fluorescence can give information about the mechanism of quenching for each species. That is, we ask are both species involved in the sensor response, to the same extent, and are the quenching mechanisms the same for the monomer and dimer? The following results are the average of data collected from six identically prepared R6G-Nafion films incubated in increasing Cu(I1) concentration solutions. Relative standard deviations for the recovered kinetic parameters are 6%. In all cases, the multifrequency traces were best modeled by double-exponential decay laws. Further, we found that the fractional concentrations of the monomer and dimer were not affected by the addition of Cu(I1). Therefore, the ground-state formation constant for the dimer is not perturbed by the presence of Cu(I1). Both fluorescence lifetimes, however, were decreased significantly in the presence of Cu(I1). SternVolmer plots (I) (not shown) for the monomer and dimer are linear. The apparent Stern-Volmer quenching constants are compiled in Table I and support a model in which the R6G species are quenched (at least partially) via a dynamic mechanism (1). In other words, Cu(I1) does not associate with either species (R6G monomer or dimer) to form a nonfluorescent complex. Again, this is not too unexpected considering that R6G and Cu(I1) are both mutually repelling cations. Instead, Cu(I1) apparently diffuses to and quenches both the monomer and the dimer species during their excited state (Table I). However, it is important to note that the apparent bimolecular quenching constants (k,) reported in Table I are only relative values. That is, we did not attempt to quantitate the amount of Cu(I1) in each film. Rather, the concentration of Cu(I1) is taken as the concentration of Cu(I1) solution in which the films were incubated. The actual Cu(I1) concentration in the films is most likely higher because of the cation-exchange ability of the Nafion. Underestimating the Cu(I1) concentration in the films leads to the overestimation of k,. As a result, the k , values are all greater than expected for a liquid-phase diffusion-controlled process (i.e., 1 x 1O'O M-l s-*). Still, comparisons of relative rates are useful here. For example, there is obviously a marked difference in rates between the quenching of the dimer and the monomer; the dimer is much more effectively quenched. In addition, the dimer in the wet film too is much more effectively quenched. These results can be explained based on a extension of the model shown in Figure 6. In the dry films, the dimer is more easily quenched because it is not paired as strongly with the sulfonate groups of the Nafion. As a result, there is a higher probability of collision of a dimer with Cu(I1). When water is added, the dimer diffuses farther from the Nafion walls while the monomer is still near. The dimer in the water pools are effectively quenched by the Cu(I1) because dimer can be
801
approached from any direction. The probability of the monomer being quenched, however, remains the same because its location remains the same (near the walls). Quenching is more ineffective near the walls. Effects of Film Preparation. Until now, all discussions have been based on experiments using solution-cast Nafion films. There has been, however, concern about the characteristics of solution-cast versus commercially available films (25). The commercial films are reported to have a higher degree of crystallinity (25). We conclude this work with a brief study on the absorbance and emission characteristics of cast and commercial Nafion films. Figure 1 shows that there is virtually no difference between the absorbance spectral contours of solution-cast and commercial Nafion for both wet and dry films. Note, however, that the commercial film absorbance, although incubated in the same concentration R6G solution, is more intense because the film is thicker (i.e., more sites for R6G). Emission spectra (results not shown) also show no differences. Both the monomer and dimer emit from the wet and dry films, and the decay kinetics follow a trend similar to the cast film results. From this, we conclude that the photophysical properties of R6G in commercial Nafion films are the same as in solution-cast f i i s and that sensors constructed from each material should .be equivalent. Only the response time of the sensor may be affected due to film thickness (i.e., diffusion through the Nafion film membrane).
CONCLUSIONS We have demonstrated that, with certain precautions, high-quality, accurate multifrequency phase and modulation data can be obtained for species a t surfaces. For good signal-to-noise (SIN > 100) characteristics, data acquisition times of about 1 min are required. By using the fiber-optic probe, we found that no biases are induced in the experimental data. We suggest that eosin isothiocyanate on controlled pore glass is a good reference material to verify that all biases have been eliminated. Although the R6G-Nafion films are not true "surfaces", it has been shown that this technique can give important information about interfaces. The results of the present study yield new insight into the "function" of the R6GNafion films as they apply to chemical sensors. For example, we believe that the mechanism of action for the previously described humidity sensor (17) involves changes in the levels of R6G monomer and dimer with added water and not simply hydrated versus unhydrated R6G. In addition, we have shown that the quenching mechanism (by Cu(I1)) within these same films is a t least partially dynamical. It also appears that the R6G dimer is more effectively quenched because of its location in the film. In contrast, the R6G monomer (preferentially located a t the interior walls or the Nafion pore) is not effectively quenched because it is shielded somewhat from the quencher. Although one could certainly suggest other models, we feel the simple models proposed here agree with past and present observations. Most recently, we have begun to study true interfaces, both simple organic surfaces (e.g., surface-immobilized cyclodextrins) and complex biological interfaces (e.g., surface-immobilized albuma and antibodies), and to determine the effects of immobilization on the chemistry as applied to fiber-optic sensors (12-16). Finally, the methodology presented here could be used to determine the transduction mechanism of any fluorescencebased chemical sensor. For example, we cite as examples those sensors based on photoinduced electron transfer (26),energy transfer (27,28),surfactant quenching (29), macromolecular ion-selective carriers (30),porous glass entrapped pH-sensitive dyes (31),Langmuir-Blodgett films (32),and membrane potential (33).
Anal. Chem. 1991, 63,802-807
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ACKNOWLEDGMENT We thank Gary Sagerman and Steve Palistrant from the fabrication of equipment necessary for interfacing our SLM 48000 MHF to fiber-optic sampling.
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RECEIVED for review November 6 , 1990. Accepted January 18, 1991. This work was generously supported by CHE8921517 awarded by the National Science Foundation and 3M, Inc. K.S.L. also acknowledges support from the 1989 ACS Analytical Division Summer Fellowship sponsored by the Pittsburgh Conference.
On-Column Sample Gating for High-speed Capillary Zone Electrophoresis Curtis A. Monnig and James W. Jorgenson* Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290
Hlgh-speed zone ekdrophoresls In a fused-slllca capillary Is described. Elevated electrlc fields and short caplllary lengths allow a mlxture of fluorescein lsothlocyanate (FITC) labeled amino aclds to be separated In tlmes as short as 1.5 8. Formatlon of the analyte zone at the head of the caplllary Is controlled by laser-Induced photolysis of a tagging reagent. Tht6 gathg procedure alkws rapkl and automated lntrodudkm of sample Into the caplllary. Ultlmately, Joule heating of the buffer IlmHs the speed and efflclency of the Separation.
INTRODUCTION Electrophoresis has been widely used for several decades as a method for separating ionized compounds. More recently, there has been growing interest in capillary electrophoresis (CE) as a general, high-efficiencymeans of separating complex mixtures. In capillary electrophoresis, the separation is carried out in a capillary tube with a typical inner diameter of 5-100 I.cm and a total length of 30-100 cm. The small radial dimensions of the capillary allow Joule heat to be dissipated efficiently, which in turn allows potentials as high as 30 kV to be applied across the length of the capillary. As a result, excellent separation efficiencies (>1 OOO OOO theoretical plates)
*
Author t o whom correspondence should be addressed. Phone number: (919)966-5071. 0003-2700/9 I /0363-0802$02.50/0
have been reported for many compounds, often in analysis times as short as a few minutes. When compared with chromatographic separation procedures, CE can offer a significant improvement in both speed and efficiency for the separation of charged species. With a typical CE instrument, separation of a mixture usually requires between 5 and 30 min. Although this time is fast relative to many competitive procedures, it is slow relative to many chemical events. As a result, CE has not been used as a method for monitoring dynamic chemical systems. To gain this capability, it is necessary to increase the speed of the analysis. One of the motivating forces behind this interest is the possibility of using such high-speed devices in a coupledcolumn multidimensional separation instrument. The resulting multidimensional instrument would have a separating power far in excess of any single-dimension instrument. However, for a coupled-column instrument to be practical, the separation in the second (or higher order) dimension should have an analysis time that is short relative to the preceding dimension. If this condition is not met, resolution in the preceding dimension will be sacrificed. Consequently, the speed of the second dimension can play an important role in determining the overall analysis time of the multidimensional instrument. To address the need for increasingly rapid CE separations, we have begun investigating a method for increasing the speed of electrophoretic separations. In this paper we will demon@ 199 1 American Chemical Society
