Anal. Chem. 1086, 58,1424-1427
1424
of the rotoreflective system for a tightly focused beam condition may lie in the dimension of the sample cell. To maintain optimal sensitivity, the path length of the cell should be no more than one-third of the system's confocal distance (22)*
The principal disadvantage of the rotoreflected beam system is that the differential thermal lens technique (2) is apparently not applicable to the present configuration. In a situation where the background signal is huge, the sensitivity enhanced may be overwhelmed by the increased background noise. The principal advantages of the rotoreflective system are experimental simplicity and the absolute sensitivity afforded. The latter is especially attractive to an analytical situation where the sample size is limited, for example, detecting minute quantities of effluents from a microbore column. The construction of a rotoreflective thermal lens/ fluorescence detection system for microbore liquid chromatography is currently under way in our laboratory.
Miyaishl, K.; Imasaka, T.; Ishibashi, N. Anal. Chem. 1982, 5 4 , 2039. Pang, T.-K. J.; Morris, M. D. Anal. Chem. 1984, 56, 1467. Leach, R. A.; Harris, J. M. Anal. Chem. 1984, 56, 1481. Nolan, T. G.; Weimer, W. A.; Dovichl, N. J. Anal. Chem. 1984, 56, 1704. Yang, Y. Anal. Chem. 1984, 56, 2336. Yang, Y.; Halrrell, R. E. Anal. Chem. 1984, 5 6 , 3002. Long, G. R.; Bialkowskl, S. E. Anal. Chem. 1984, 56, 2806. Pang, T.-K. J.; Morris, M. D. Appl. Spectrosc. 1985, 39, 90. Jansen, K. L.; Harris, J. M. Anal. Chem. 1985, 5 7 , 1698. Jansen, K. L.; Harris, J. M. Anal. Chem. 1985, 5 7 , 2434. Berthoud, T.; Delorme, N.; Mauchlen, P. Anal. Chem. 1985, 57, 1216. Yang, Y.; Hall, S. C.; De La Cruz, M. S. Anal. Chem. 1988, 58, 758. Harris, J. M.; Dovichi, N. J. Anal. Chem. 1980, 52, 695A. Jenkins, F. A.; White, H. E. Fundamentals of Optics, 3rd ed.; McGrawHill: New York, 1967. Whinnery, J. R. Acc. Chem. Res. 1974, 7 , 225. Carter, C. A.; Harris, J. M. Appl. Opt. 1984, 23, 476. Fang, H. L.; Swofford, R. L. Ultrasensitive Laser Spectroscopy; Academic Press: New York, 1983; p 175. Carter, C. A.; Harris, J. M. Anal. Chem. 1984, 56, 922. Sepaniak, M. J.; Vargo, J. D.; Kettler, C. N.; Maskarinec, M. P. Anal. Chem. 1984, 56, 1252. Nolan, T. G.; Hart, B. K.; Dovichi, N. J. Anal. Chem. 1985, 5 7 , 2703. Oriel Optlcs & Filter Catalog, 1985; Vol. 111.
LITERATURE CITED (1) Long, M. E.; Swofford, R. L.; R. L.; Albrecht, A. C. Science (Washington, D . C . ) 1978, 791, 183. (2) Dovlchi, N. J.; Harris, J. M. Anal. Chem. 1980, 52, 2338. (3) Dovichi, N. J.; Harris, J. M. Anal. Chem. 1981, 53, 106. (4) Morl, K.; Imasaka, T.; Ishibashi, N. Anal. Chem. 1982, 5 4 , 2034.
RECEIVEDfor review January 21,1986. Accepted February 27,1986. This research was supported by grants from Loyola University of Chicago Summer Research Award, Research Stimulation Funds, and Small Research Grant.
Three-Component Determinations Using Fluorescence Anisotropy Measurements and Wavelength Selectivity Frank V. Bright' and Linda B. McGown* Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74078
Fluorescence anlsotropy measurements at a serles of excltatlon wavelengths were used to simultaneously determine three anthracene derlvatives In mixtures containlng from one to three of the derivatlves In frozen glycerol solutlons. The results are compared wlth results obtained uslng nonpolarization measurements of fluorescence intensity at the same serles of excltatlon wavelengths. Average error magnitudes obtained for the determlnatlons uslng anlsotropy measurements were signlficantiy smaller than those obtalned for the nonpoiarizatlon determlnatlons, indicating the superior selectivity achieved between these three components uslng the anlsotropy approach.
The applications, instrumentation, and theory of fluorescence anisotropy and polarization measurements have been described in detail elsewhere (1-11) and are based on the photoselection of fluorescent species as a function of their molecular orientations relative to polarized exciting light. Anisotropy measurements determine the average angular displacement of a fluorescent species during the lifetime of its excited state. If a sample is excited by vertically polarized light at excitation wavelength A,,, the corresponding anisotropy (r(Aex)) is given by Present address: Department of Chemistry, Indiana University, Bloomington, IN 47405. 0003-2700/86/0358-1424$01.50/0
where Ili (Aex) is the fluorescence intensity observed through a vertically oriented emission polarizer and IL(Aex) is the fluorescenceintensity observed through a horizontally oriented emission polarizer. The anisotropy is thus a measure of the ratio of the polarized emission and the total fluorescence intensity. For a mixture of n independent, noninterfering emitters, the anisotropies are linearly additive and take the form n
where f i is the fractional contribution of component i to the total fluorescence emission intensity and r, is the anisotropy of component i. Excitation rather than emission anisotropies are generally determined because excitation spectra often involve transitions to energy levels higher than the first singlet (Sl).The higher levels have different angular displacements, resulting- in different anisotropies (7). Emission, on the other hand, is almost exclusively from the S1level so that emission anisotropy spectra are usually flat and featureless (7, 10). For dilute solutions in frozen glasses such as propylene glycol at -70 "C or glycerin at -10 "C, the observed anisotropies are essentially devoid of all depolarization processes (energy transfer, Brownian rotation, and rotational diffusion), and are denoted by ro. 0 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986 1
Results are shown here for the simultaneous determination of 9-phenylanthracene (9 PA), 9,lO-diphenylanthracene (9,lO DPA), and 9-vinylanthracene (9 VA) in frozen solutions containing one or more of these compounds, all treated as three-component mixtures, using measurements of ro a t a series of excitation wavelengths. Results are also shown for the analysis of the same solutions at the same excitation wavelengths using nonpolarization intensity measurements t o achieve wavelength selectivity alone.
EXPERIMENTAL SECTION Materials. The reagents 9-phenylanthracene(Pl, 800-4,98%), 9,lO-diphenylanthracene(D20,500-1,99%),and 9-vinylanthracene (V170-8,97%)were all purchased from Aldrich and recrystallized once from absolute ethanol, (U.S. Industrial Chemical Co.). Glycerin was purchased from Fisher and was distilled at reduced pressure (12) to minimize impurities. Standard solutions of the individual compounds (1.00 mM) were prepared by weighing the appropriate amount of each compound, diluting to 100 mL with ethanol, and sonicating for 30 min. Mixtures were prepared by warming the glycerin solution in an oil bath to 80 *C and pipetting 3.00 mL of glycerin into individual quartz cuvettes. The appropriate volume of each standard was then added to each cuvette, and the cuvettes were allowed to warm to 80 "C for 10 min to evaporate residual ethanol solvent, resulting in a total volume of 3.00 mL in each cuvette. The total fluorophore concentration in each cuvette was kept below 1/.LMto minimize energy transfer effects. Data Collection. All fluorescence measurements were made with an SLM 4800s spectrofluorometer (SLM Instruments, Inc., Urbana, IL) with a 450-W Xe arc source and Hamamatsu R928 photomultiplier tube detection. Glan-Thompson calcite prism polarizers were positioned in the excitation and emission channels in an L configuration for polarization and anisotropy measurements (10). Sample chamber temperatures were maintained at -10 0.01 OC, using a Haake A81 temperature control unit to freeze the glycerin solutions into a solid glass, thereby eliminating rotational diffusion and Brownian rotation effects. Dry N2was used to flush the sample chamber and minimize condensation on both the sample cuvettes and instrument optics. The excitation monochromator was scanned at 1-nm increments between 300 and 400 nm, and fluorescence emission was monitored a t 420 nm for all fluorescence spectra. Slit settings were 16 nm and 4 nm for the excitation monochromator entrance and exit, respectively, 4 nm for the modulation tank exit, and 16 nm for both the emission monochromator entrance and exit. The fluorescence intensities were acquired by an interfaced Apple 11+ microcomputer and stored on floppy disk for further data analysis. The blank contribution due to glycerin was negligible, so no correction to the measured fluorescence intensities was necessary. All spectra (polarization and nonpolarization) were recorded by using the "10 average" mode in which an average value is obtained by integration of 10 samplings over approximately 3 s, performed internally by the spectrofluorometer. The excitation spectra for all anisotropy studies were recorded at four different polarizer settings to correct for the polarization-dependent response of the monochromators. The spectral intensities corresponding to each of the four excitation spectra are represented Iw, Iw, and I" where the first subscript represents the as IVV, orientation of the excitation polarizer and the second represents the orientation of the emission polarizer (V = vertical and H = horizontal). The fluorescence anisobropy spectra (rovs. Aex) are calculated by using the following relationship: ro(Xex) = Uvv - CIVH)/UW+ 2CIw) (3) where the C term is a correction factor for the varying monochromator response and is defined as IHV/I". Data Analysis. Fluorescence intensity data taken from the acquired spectra at the selected wavelengthswere entered by hand into an Apple IIe microcomputer. Two different sets of intensity data were employed for data analysis, including the set acquired without polarizers and the set from the anisotropy spectra. In both cases, a series of simultaneous equations were generated and solved for the analytical concentrations of the individual com-
*
1425
4 N
. . .. ." .... .. :....
.-1
,
-:: ,... .... 1;: .. ..' '320
330
. .. . .. ., . '
340
1
I
I
350
360
370
380
390
400
EXCITATION WAVELENGTH (nm)
Flgure 1. Steady-state fluorescence excitation spectra for 9 PA (A), 9,lO DPA (B), and 9 VA (C); emission wavelength = 420 nm.
ponents in each mixture. The augmented matrices generated for the three-component system take the form in eq 4, where Ri is
either the anisotropy or the molar fluorescence intensity at each excitation wavelength i (for i = 1to n) for the pure species and Riis the observed fluorescence intensity or anisotropy for the mixture at excitation wavelength i. The f term represents either the analytical concentration of each species (for nonpolarization data) or the fractional contribution that each species makes to the total fluorescence intensity (for anisotropy data). Square matrices (n = 3) were solved by Gaussian elimination with scaled partial pivoting, and overdetermined matrices (n > 3) were solved by a least-squares Gauss-Newton iterative procedure (13). Concentrations are not directly determined by using the anisotropy spectral data, since anisotropy is an intrinsic characteristic that depends on the relative fractional intensity contributions of the components. The concentration of each component (CWmp) was calculated from the anisotropy data by multiplying the fractional contribution of the component (fcomp) by the total and nonpolarized fluorescence signal of the mixture (Imixture) dividing by the molar fluorescence intensity of the component (fcomp)
Ceomp
= fcomJmixture/fcomp
(5)
Solutions containing only one or two of the three components were treated as three-component mixtures to determine the ability of each approach to detect the absence of components.
RESULTS AND DISCUSSION Figures 1and 2 show the nonpolarization and the anisotropy excitation spectra, respectively, for 9 PA, 9,lO DPA, and 9 VA. The inversion of the anisotropy values for 9 VA and 9,lO DPA a t 378 nm (Figure 2) adds to the selectivity of the anisotropy approach. Therefore, excitation wavelengths between 370 and 390 nm located on either side of the 378-nm crossing point were used for all results presented. Since wavelength optimization was not used for either approach, there is a possibility that a bias toward one or the other of the approaches was introduced by the choice of wavelength. However, in both approaches the wavelength range used (with as many as 20 wavelengths) included crossover points between the spectra of the individual components. Table I shows the analytical concentrations and fractional intensity contributions of the three components in the 16
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7,JUNE 1986
1426
0.40 ,
Table 11. Statistical Information for the Plots of Experimentally Determined Concentration vs. Actual Analytical Conceptration for the Solutions Described in Table I
.. ..'..' *
..I.
I
..a
0.351
....
....... .........................
. .... .. .......
....:::::.... ...... . . ....(..'
:I*.
w
B ,.." ."" ........ . .... .. .'C ....... ,.a*.
I
2 0
H
8
.......
0.20 ::.:
0.15
. ........ . ... . . ......... .....
I
1
1
1
~
Table I. Analytical Concentrations and Fractional--Intensity Contributions of Individual Components in the Solutions Used in this Study
soh 1 2
3 4 5 6 7 8 9 10 11 12
13 14 15 16
9 PA 0.026 (0.050) 0.464 (0.900) 0.026 (0.050) 0.129 (0.250) 0.129 (0.250) 0.258 (0.500) 0.171 (0.333) 0 (0)
0.258 (0.500) 0.258 (0.500) 0.386 (0.750) 0.386 (0.750) 0 (0) 0 (0) 0 (0)
0.515 (1.000)
concn,nWM 9,lO DPA 0.049 (0.050) 0.049 (0.050) 0.883 (0.900) 0.245 (0.250) 0.491 (0.500) 0.245 (0.250) 0.327 (0.333) 0.491 (0.500)
9 VA
0 (0)
0.468 (0.900) 0.026 (0.050) 0.026 (0.050) 0.260 (0.500) 0.130 (0.250) 0.130 (0.250) 0.173 (0.333) 0.260 (0.500) 0.260 (0.500)
0.491 (0.500)
0 (0)
0 (0)
0.130 (0.250)
0.245 (0.250) 0.736 (0.750)
0 (0)
0 (0)
0.981 (1.000) 0 (0)
0.130 (0.250) 0.520 (1,000)
species
slopen
intercept"
rb
SEEc
9 PAd 9 PAe 9 PAf 9 PAS 9,lO DPAd 9,lO DPAe 9,lO DPAf 9,lO DPAg 9 VAd 9 VA' 9 VAf 9 VAg
0.871 (0.092) 0.920 (0.074) 0.985 (0.014) 0.990 (0.006) 0.900 (0.053) 0.964 (0.044) 0.972 (0.018) 0.980 (0.007) 0.812 (0.079) 0.727 (0.091) 0.964 (0.021) 0.985 (0.009)
0.015 (0.024) 0.005 (0.020) 0.002 (0.004) 0.001 (0.002) 0.064 (0.024) 0.041 (Q.020) 0.005 (0.008) 0.006 (0.003) 0.025 (0.017) 0.034 (0.020) 0.010 (0.005) 0.003 (0.002)
0.9297 0.9573 0.9986 0.9997 0.9765 0.9855 0.9976 0.9997 0.9401 0.9061 0.9965 0.9993
6.37 5.13 0.98 0.41 6.68 5.60 2.27 0.82 4.95 5.71 1.36 0.69
"Absolute error for each term is in parentheses. bCorrelation coefficient. Standard error of estimate ( N O 2 wM). dThree wavelengths at 372, 377, and 380 nm (no polarizers). eSeven wavelengths at 370, 372,374,377,379, 381, and 383 nm (no polarizers). 'Three wavelengths at 372, 377, and 380 nm (anisotropy). gSeven wavelengths at 370, 372, 374, 377, 379, 381, and 383 nm (anisotroov). Table 111. Total Recovery for Each Component from the 16 Solutions Determined Using Anisotropy Measurements at Three and Seven Excitation Wavelengths
9 PA 3b found actual
3.011 3.032
total component concn, pMn 9,lO DPA 9 VA 7b 3* 76 36 76
3.023 3.032
re1 error -0.69% -0.30%
5.155 5.233 -1.5%
5.228 5.233
2.543 2.522 2.513 2.513
-0.10%
1.2% 0.48%
The sum of the concentration of the component in all 16 solutions, in cuvette. bThe number of excitation wavelengths used. 30r
0 (0) 0 (0)
Concentration in cuvette with fractional intensity contribution in Darentheses. individual solutions. Table I1 shows the statistical information for plots of the experimentally determined concentrations vs. the actual analytical concentrations (Table I) for each of the two approaches for determinations using data generated at three excitation wavelengths and at seven excitation wavelengths. Data sets generated using from 3 to 20 wavelengths in the range of 370-390 nm gave similar results. The total recoveries of each component from all 16 solutions determined by the anisotropy approach using measurements a t three and a t seven excitation wavelengths are shown in Table 111. Good accuracies were achieved even for the determination of components with fractional intensity contributions as low as 5%, as well as for the analysis of solutions in which one or two of the components were absent in the three-component system. The average magnitudes of the absolute determination errors are shown in Figure 3 as a function of the number of excitation wavelengths used. The average error magnitudes shown in Figure 3 were calculated by taking the absolute value of the absolute error for the determination of each component in each solution, summing over all three components in all 16 solutions, and dividing by 48 to get the average value. The average error magnitudes decreased as the number of equations approached 10 for both the anisotropy and the nonpolarization determinations. However, the average error mag.
201
$
15-
a
z
$
105
I
0
5
---
1
10
I
1
15
20
NUMBER OF EXCITATION WAVELENGTHS
Flgure 3. Average absolute error magnitudes for the determination of the three components in the 16 solutions described In Table I vs. the number of excltation wavelengths used, for the anisotropy (m)and the nonpoiarization approaches (0).
nitudes were substantially smaller for the anisotropy approach, indicating that this approach provides better selectivity between the three components used in this study. Registry No. 9 PA, 602-55-1; 9,lO DPA, 1499-10-1; 9 VA, 2444-68-0.
LITERATURE CITED (1)
Jablonski, A. Bull. Acad. Pol. Scl. 1960, 8, 259.
(2) Perrin, F. Ann. Phys. (Paris) 1929, 12, 189. (3) Weber, G. Biochemlstiy, 1952, 57, 145. (4) Weber, 0. Blochemistry, 1952, 51, 155. (5) Weber, G. Biochemistry, 1960, 75, 345.
Anal. Chem. 1986, 58, 1427-1430 Weber, 0. Biochemistry, 1960, 51, 335. Valeur, B.; Weber, G. Photochem. Photobiol. 1977, 25, 441. Chuang, T. J.; Eisenthal, K. B. J . Chem. Phys. 1972, 5 7 , 5094. Weber, G. I n Fluorescence and Phosphorescence Analysis ; Hercules, D. M., Ed.; Interscience: New York, 1966; Chapter 6. (IO) Lakowicz, J. R. Princlples of Fluorescence Spectroscopy; Plenum: New York, 1983; Chapter 5. ( 1 1 ) Jameson, D. M.; Weber, G.; Spencer, R. D.; Mitchell, G. Rev. Sci. Instrum. 1978, 49, 510.
1427
(12) Perrin, D. D.; Armarego, W. L. F.; Perrin, D. R. Purification of Laboratory Chemicals; Pergamon: New York, 1960; p 275. (13) Bright, F. V. Ph.D. Dissertation, Oklahoma State University, Stlllwater, OK, July 1985.
for review September
199
1985* Accepted February
3, 1986.
Polymer Modification of Fiber Optic Chemical Sensors as a Method of Enhancing Fluorescence Signal for pH Measurement Christiane Munkholm and David R. Walt* Max Tishler Laboratory for Organic Chemistry, Tufts University, Medford, Massachusetts 02155
Fred P. Milanovich Lawrence Livermore National Laboratory, University of California, Livermore, California 94550
Stanley M. Klainer
ST&E Technical Services, Inc., 20 Belinda Court, S a n Ramon, California 94580
We have prepared a pH fiber optlc sensor based on fiuorescence intensity. Fiuoresceinamine is incorporated into an acryiamide-methyienebis(acryiamide) copolymer that Is attached covalently to a surfacemodifiedglass fiber via thermal or photopolymerization. The sensor gives instantaneous responses and reversible measurements over the pH range of 4.0-8.0 with slgnal-to-noise ratios typically 275/1. The resutls indicate that specific fiber optic chemical sensors can be mlniaturlzed and stlli retain sufficient signal intenslty and stability.
Small-diameter optical fibers offer the potential for use as continuous sensing devices (1-5). Previous sensors have been prepared that use a membrane, tubing, or hollow fiber superimposed around the end of an optical fiber which contains a species-specific reagent (1-3, 6, 7). The limitations of previous sensors (1,3 , 4 , 7 , 8 ) have been (1)the small signal arising from attachment to a limited fiber surface area, (2) the cumbersome task of assembling individual membrane fiber sensors, (3) the long response time of the fiber due to diffusional limitations of the membrane, and (4)the degradation and lack of specificity of the sensing material. In this paper we report the chemical modification of small-diameter optical fibers, which results in an enhanced signal and which overcomes many of these previous limitations. For an optical fiber to serve as a sensor a reagent phase must be in the vicinity of the distal end of the fiber. Mere attachment of a fluorescent compound to the surface of an optical fiber results in no detectable signal due to the limited surface area available. Our approach is based on the covalent attachment of a polymer to each reactive site of the surface. This approach is similar to the polymer modifications that have been performed on electrodes (9-12). The polymer serves to increase the surface area and results in multiple sites of attachment for the fluorescent species. Since this method does not require a membrane, the signal response is rapid. 0003-2700/86/0358-1427$01.50/0
EXPERIMENTAL SECTION Materials used included ammonium persulfate (Bio-Rad Labs) and riboflavin and [y-(methacryloxy)propyl]trimethoxysilane (Pharmacia). Ammonia and argon were from Matheson Gas Co., and n-hexane (class 1B) was acquired from Fisher Scientific Co. All other materials were purchased from Aldrich Chemical Co. All reagents were used without further purification. Corning Core Guide glass/glass optical fibers of nominally 100/140 (hm/hm) diameter were cut into l-m lengths, terminated with amp connectors on one end, and both ends were hand polished. The polished distal end was protected with a retractable capillary tube during all operations. Apparatus. All measurements described here were performed on the instrument shown schematically in Figure 1. This instrument has been described previously (13);therefore, only a brief description is presented here. Light of appropriate wavelength is directed into an optical fiber with moderate focusing. In doing so, it passes through either a dichroic mirror or a front surface reflector with a small perforation (as shown in Figure 1). The returning fluorescent signal exits the same terminus of the optical fiber at the numerical aperture (NA) of the optical fiber. This returning (red-shifted) light is deflected by the dichroic or front surface mirror and subsequentlydirected into a spectrometer for analysis. Fibers used in this work had an NA of 0.28. Consequently, light exited these fibers with a diverging half-angle (approximately) of 16.3" (Figure lb). The reactor used for the polymerizations is shown in Figure 2; the method of N2 delivery was adapted from a manifold design of Schwartz (14). Surface plasma chemistry was performed with an electrical chemical induction coupled glow discharge system (Figure 3). The Pyrex glass chamber was handmade; the luminous tube transformer was from Acme Electrics, Cuba, NY. Procedure. Surface Silanization with [y-(A4ethacryloxy)propyl]trimethoxysilane. A 2 % solution was prepared according to the method of the manufacturer (Pharmacia Fine Chemicals) by mixing 100 r L of [y-(methacryloxy)propyl]trimethoxysilane in water adjusted to pH 3.5 and then stirring for 15 min. The fibers were submerged in this solution and soaked for 1 h, then rinsed with water and dried in a desiccator for 1 h. Surface Silanization with (Aminopropy1)triethoxysilane.A 10% aqueous solution of (aminopropy1)triethoxysilane was ad@ 1986 American Chemical Society
