Anal. Chem. 1988, 58, 2870-2872
Molecular Fluorescence in Thin Liquid Films Sir: Aqueous surfactant solutions are known to form thin films when suspended on a suitable frame. Mysels produced vertical films by submerging a rectangular frame into a solution and then withdrawing it partially (I). Frame size was 3 cm X 2 cm, and initial film thickness was 0.2-10 pm, depending upon the velocity of pull out. Once formed, the film thins by drainage and evaporation to an equilibrium thickness in the range of 100-1000 A. Theory for the rate of thinning and eventual rupture of such films is well-documented (2-4). Several qualities of thin liquid films suggest that they might be excellent sample cells for the measurement of molecular fluorescence. The extremely small path length across the film gives rise to sample volumes less than 10 KL. Furthermore, this amount of sample may be distributed over an area of 5 cm2or more. These size constraints should reduce the prefilter effect observed in standard quartz cuvettes. Absolute limits of detection should also be well below those obtained from conventional sample cells. In conventional fluorescence cuvettes, the excitation beam passes through at least two interfaces: one solid-gas and one liquid-solid. The emitted light also passes through two such interfaces. The resulting scattered light may cause a small reduction in fluorescence intensity as well as an increase in background interference. Only two interfaces are present with thin films, both liquid-gas, so scattered light should be reduced. Any fluorescence background from the quartz cuvette should also be eliminated (5). Finally, a wide variety of molecules are soluble in aqueous surfactant solutions. Nonpolar molecules are soluble if the concentration of surfactant is above the critical micelle concentration (cmc). Polar molecules do not require high surfactant concentrations and are more easily analyzed. EXPERIMENTAL S E C T I O N Instrumental Methods. Absorption measurements were made with the Hewlett-Packard Model 8450A diode array UV-vis spectrophotometer. Molecular fluorescence was measured with the Aminco-Bowman spectrophotofluorometer. Excitation and emission slits were 2.0 mm. The light source was a 150-W xenon arc lamp, Signal from the photomultiplier tube was read directly from an Aminco ratio photometer, or from a strip chart recorder. A syringe pump (Sage Instruments, Model 341) was used for the flow-throughsystem. With a 20-mL syringe, flow rates from 0.2 to 6 mL/min were obtained. Methodology. Pyrene, quinine sulfate monohydrate, and cetyltrimethylammonium bromide (CTAB) were obtained from Aldrich Chemical Co. p-Aminobenzoic acid was obtained from Fisher Scientific. All solutions were made from triply distilled water. Stock solutions of pyrene (1000 +g/mL) were prepared in a methano1/0.005 M CTAB solution (2080 v/v) due to solubility restrictions. Quinine stock solutions (loo00 pg/mL) were prepared in solvent that was 0.05 M H2S04 and 0.001 M CTAB. p Aminobenzoic acid stock solutions (loo00 pg/mL) were prepared in 0.001 M CTAB. Higher surfactant concentrations, 0.02 M CTAl3, were used for the flowing system. At this concentration, the film was less sensitive to changes in flow rate. Standard quartz cuvettes (1 cm x 1 cm) were used for conventional absorption and fluorescence measurements. Thin liquid films were suspended on a round wire loop made from oxygen-free high conductivity copper. The loop diameter was 1.90 cm, while the wire itself had a 1 mm diameter. To reduce direct scatter in the fluorescence measurements, the loop was situated at an angle of approximately 40' with respect to the excitation beam, and 50° with respect to the measured emission beam. Scatter was further reduced by situating the loop such that the emission was monitored from the backside of the film. Liquid films were formed by raising a small beaker of solution over the loop and lowering it slowly. Fluorescence (or absorbance) at a fixed wavelength was then measured with respect t o time (Table I).
Table I. Wavelengths for Fluorescence Measurements compound
p-aminobenzoic acid pyrene
332 295 322
nm 450 345
Table 11. Analytical Figures of Merit LOD,
compound quinine pyrene
cuvette thin film cuvette thin film
1 30 1
3 0.1 3 0.02 2 0.1
In the flowing system, a hypodermic needle was placed at the top of the loop. The needle was connected to a syringe, and a syringe pump, by a length of small internal diameter flexible tubing. The film was initially suspended on the loop by the method described above, and flow through the needle was adjusted to replenish any loss of solution due to drainage or evaporation. At the appropriate flow rate, "flowing" films persisted until the sample reservoir (syringe) was depleted (up to 1 h). RESULTS AND DISCUSSION Determination of Film Thickness. Film thickness was determined by absorbance measurements and the implementation of Beer's law. Calibration curves were measured for pyrene in a cuvette and in a thin film. The thickness of the film was calculated from the slopes of the plots by using the equation ~ T = F ~ c ( ~ T F / ~ c )
where hTF is the thickness of the film in cm, hc is the path length in the cuvette (1cm), mTFis the slope of the calibration curve with the thin film, and mc is the slope of the calibration curve with the cuvette. Appropriate blanks were subtracted from each measurement, and the molar absorptivity coefficient for pyrene was assumed to be constant with time (regardless of cell type). Thinning of the film was calculated as a function of time (Figure 1). Initial thicknesses were consistent with those in ref 1. Film lifetimes were on the order of 10 s, with rupture resulting from excessive drainage and evaporation. Static Fluorescence Measurements. The fluorescence of pyrene, quinine, and p-aminobenzoic acid was measured at fixed wavelengths as specified in Table I. Fluorescence was measured as a function of time (Figure 2). Analytical calibration curves were plotted for each compound at time zero (the time corresponding to initial suspension of the film). Figures 3 and 4 show analytical calibration curves for quinine and pyrene in standard cells and in thin liquid films. Analytical figures of merit are reported in Table 11. The similarity between Figures 1 and 2 suggests that fluorescence decreases with film thickness. Optimum sensitivity is obviously realized at maximum film thickness (time zero). Sample volumes are calculated by multiplying film thickness by the area of the loop. Absolute limits of detection are calculated by using the average sample volume at time zero, 3.6 wL. Those found for the thin film are at least an order of magnitude lower than those for conventional cells. This may be attributed to smaller sample volume, lower light scatter in the sample compartment, and more efficient excitation due
0003-2700/86/0358-2870$01.50/00 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58,NO. 13, NOVEMBER 1986
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Flgure 1. Thickness of a thin liquid film as a function of time. 14
Flgure 2. Relative fluorescence of pyrene (20 pg/mL) in a thin film as a function of time: A, 322 nm, A,, 371 nm.
U i v e l e n g t h (nml 10'
Flgure 5. Fluorescence spectra of 20 Mg/mL pyrene in 0.02 M CTAB: (A) in a standard 1-cm cuvette, and (B) in a thin film with a flow-through rate of 0.5 mL/min. The sensttivity for part B is 10 times greater than that for part A.
-: L PI
Flgure 3. Analytical caiibration curves of quinine in a standard 1-cm cuvette (asterisk) and in a thin film (circle).
to the "spreading" of the small sample volume over a relatively large area. The linear dynamic range, LDR, is often greater with the thin film than with conventional cells. This is probably due to the smaller path length in the film. Higher concentrations are necessary to observe an appreciable prefilter effect over such a small distance. Any postfilter effects similarly should be reduced in thin films. For the calibration curves of pyrene, linearity is lost a t an unusually low concentration. This may be attributed partially to pyrene excimer formation in solutions with concentrations
above 5 pg/mL. In these solutions a strong excimer emission is observed in the region of 450 nm (Figure 5). The ratio of excimer to monomer fluorescence was found to increase with increasing pyrene concentration. The ratio was found to decrease with increasing surfactant concentration. Some variation was inevitable in the measurement step. Initial film thicknesses varied by as much as 6%, when calculated from repeated absorbance measurements. Consequently, the fluorescence signal a t time zero had a relative standard deviation of up to 6%, for solutions with analyte concentrations greater than 10 times the LOD. Multiple measurements were quick and easy to obtain, so five to ten measurements were made for each solution. Average signals were used in the calibration graphs. At concentrations nearer the LOD, a 10% relative standard deviation was typical. Film lifetimes also had some variation. For the 0.005 M CTAB solutions, the average film lifetime was 8.6 f 0.8 s. Lifetimes for the 0.001 M CTAB solutions were a bit shorter, 6.0 i~ 0.8 s. Twenty lifetimes were taken for each solution. Fluorescence in "Flowing" Films. Although absolute detection limits are low in thin films, large volumes (5 mL) of sample are required for initial suspension of the film. For this reason, better methods for depositing samples into the filmme necessary. One such method involves flowing a sample solution through the film a t a constant rate. At flow rates of about 0.5 mL/min, film lifetimes may exceed 1h, depending
Anal. Chem. 1986, 58, 2872-2874
on reservoir size. Figure 5 shows a comparison of the fluorescence spectrum of pyrene in a cuvette to that of the same solution when flowed through a thin film. This suggests that molecular fluorescence in thin liquid f i i s may be a useful as a detection technique for liquid chromatography or flow injection analysis. Current research is aimed in this direction.
Registry No. CTAB, 57-09-0; pyrene, 129-00-0; quinine, 130-95-0;p-aminobenzoic acid, 150-13-0. LITERATURE CITED (1) Mysels, K. J. J . Phys. Chem. 1964, 68, 3441-3448. (2) Radoev, B. D.; Dimitrov, D. S.;Ivanov, I.B. Colloid Polym. Sci. 1974, 252,50-55.
(3) Ivanov, I. B.; Dirnitrov, D. S. Colloid Polym. Sci. 1974, 252, 982-990. (4) Manev, E. D.: Vassilieff, Chr. St.; Ivanov, I. 6.Colloid Polym. Sci. 1976, 254, 99-102. (5) Parker, C. A. Photoluminescence of Solutions: Eslevier: New York, 1968; Chapter 5.
Bradley T. Jones James D. Winefordner* Department of Chemistry University of Florida Gainesville, Florida 32611
RECEIVED for review March 25,1986. Accepted June 25,1986. This research was supported by NIH-5R01-GM11373-22.
Polyacrylamide/Graphite and Polyacrylamide/Titanium Dioxide Gel Electrodes Sir: Polyacrylamide (pacr) gels have attractive properties that are the basis for several important applications in bioanalytical chemistry. For example, they provide a hydrophilic environment with a controllable pore size that allows for the entrapment and immobilization of bioplymers in their active forms. Pacr gels have long been used as matrices for gel electrophoresis and gel permeation chromatography. Recently, we described the construction of an enzyme electrode, based on the ideas of Hill and co-workers ( I ) , that consisted of an enzyme/mediator system entrapped in a pacr interface between a carbon support bed and the solution ( 2 ) . These pacr-modified electrodes mediated the direct and specific amperometric oxidation of glucose. Others ( 3 )have studied pacr-modified electrodes, and a variety of applications based on the electrochemically driven swelling of polyelectrolyte gels can be envisioned (4). Thus these electrodes show promise as permeable, multicomponent interfaces between an electrode substrate and solution. The purpose of the work described here was 3-fold: first, to study charge transport through the ferrocene/graphite/pacr gel composite; second, to document preliminary investigations of the photoactivation process occurring in the ferrocene/Ti02/pacr gel; and third, to examine the analytical utility of the pacr electrode. EXPERIMENTAL SECTION Reagents and Apparatus. All experiments (unless otherwise noted) were carried out in pH 7 Sorenson phosphate buffer solutions (0.0667 M) and potentials are referred to the Ag/AgCl reference electrode. Filtered distilled water (Milli-Q System, Millipore) was used to prepare solutions. Ferrocene was used as received from ROC/RIC. Acrylamide (Aldrich gold label) and riboflavin (Merck & Co., Inc.) were used to prepare the gels. Linear sweep and cyclic voltammetry and differential pulse voltammetry were done on a BAS-100 electrochemical analyzer. Chronocoulometry was performed with a Princeton Applied Research Model 173/ 175 potentiostat/programmer combination. Charge data were taken in digital form using a Nicolet Model 200 oscilloscope. Electrode Fabrication. Photopolymerization of acrylamide has been described previously ( 2 , 5 ) . The photopolymerization is terminated before the gel becomes firm, and then the gel is mixed with powdered graphite (Union Carbide Corp., Grade 38) to yield a mixture which is 40% by weight graphite with the consistency of chewing gum. This composite is then packed into a strip of reticulated vitreous carbon (RVC) and mounted into a Teflon sleeve to give the gel/graphite interface a geometrical surface area of ca. 0.28 cm2. Colloidal Ti02 was prepared in a manner similar to that described by Duonghong et al. (6). One milliliter of Ti(OCH(CH&),
Table I. Chronocoulometry of Ferrocene/Graphite/Pacr Gels" w t % Fc
10.5 14.2 18.5 22.7
109D1~2c/mo1 cm-2 s-l/2 10.lb
a Cottrell slopes for applied potential steps from 0.0 to 0.5 V in pH 7 Dhowhate buffer. bRelativestandard deviation = 10%.
was added to 20 mL of 2-propanol. A 0.2-mL aliquot of this solution was slowly injected into 20 mL of pH 1 HCl solution. The resulting suspension showed an absorption band at 340 nm in agreement with the literature (6). The precipitate was collected, dried at 120 OC, pulverized to a fine powder, and used as above, in place of the graphite, to make the Ti02 gel electrodes. An Aminco-Bowman spectrophotofluorometer, Model No. 4-8202, was used to measure the wavelength response of these electrodes. A spectroline, Model llSC-2, Hg lamp was used to activate the electrodes. For the studies described below, the electrodes were baked in a drying oven at 125 "C until they appeared to be completely dehydrated. Of course this step was omitted in the previous study of pacr/enzyme electrodes (2). After dehydration, the electrodes were loaded by application of an aliquot of solution containing the analyte, which was rapidly imbibed by the interface. The loaded electrodes were then immersed in the pH 7 buffer and subjected to electrochemical analysis.
RESULTS AND DISCUSSION Chronocoulometry of Ferrocene/Graphite/Pacr Gels. The mechanism of charge transport through a gel matrix containing mediator sites or redox couples is a complex process. Electron exchange reactions along with incorporation of aqueous electrolyte and motion of the pacr matrix must occur. Electrolysis results in huge volume changes, which must be considered in a practical or theoretical analysis. Accordingly, it is difficult to predict a priori the expected time dependence of the chronoamperometric current or the possible role of finite diffusion limitations of the gel matrix. Table I gives chronocoulometric Cottrell slope data for electrodes in which solid ferrocene is dispersed in the gel matrix. The composition range encompasses that employed in the enzyme electrodes previously described (2). The chronocoulometric data displayed linear Anson slopes of Q
C 1986 American Chemical Society 0003-2700/86/0358-2872$01,50/0