55
Anal. Chem. 1985, 57,55-59
the source of the spectral sensitivity dip near the absorption minimum of nile blue is clearly incorrect; our concentration study shows that the error arises from aggregation phenomena rather than transmission.
ACKNOWLEDGMENT We thank D. G. Taylor for valuable suggestions on the beam switcher design and M. Grubb for wiring some of the circuits. J.N.D. also thanks R. A. Keller for his hospitality at Los Alamos National Laboratory where part of this paper was written during a Sesquicentennial leave from the University of Virginia. Y
LITERATURE CITED Parker, C. A. “Photolumlnescence of Solutions”; ElseCler: New York, 1968. Caivert, J. 0.; Piis. J. N., Jr. “Photochemlstry”; Wiley: New York, 1966. Heard, H. 0 . “Laser Parameter Measurements Handbook”; Wiley: New York. 1968. Zalewski, E. F. “Optical Radiation Measurements”; Mielenz, K., Ed.; Academic Press: New York, 1982; Vol. 3. Zalewski, E. F.;Geist, J.; Veiapoldl, R. A. Proc. ASTM Symp on New Directions in Molecular Lumlnescence, 1983. Gelst, J.; Zalewski, E. F.; Schaefer, A. R. Appl. Optics 1980, 19, 3795. Zalewski, E.; Tufino, M. Proc. SPIE-Int. SOC. Opt. Eng. 1981, 1, 308. Carreras, C.; Corrons, A. Appl. Opf. 1981, 10, 1174. Zalewskl, E. F.; Duda, C. R. Appl. Opt. 1983, 2 2 , 2867. I
(10) Demas, J. N.; Bowman, W. D.; Zaiewski, E. F.; Velapoldi, R. A. J. Phys. Chem. 1981, 8 5 , 2766. (11) Taylor, D. 0.; Demas, J. N. Anal. Chem. 1979, 5 1 , 71 (12) Taylor, D. G.; Demas, J. N. Anal. Chem. 1979, 5 1 , 71j. (13) Demas, J. N. “Photoluminescence Spectrometry”; Mielenz, K., Ed.; Academic Press: New York, 1982. (14) Weber, G.; Teale, F. W. J. Trans. Faraday SOC. 1957, 53, 646. (15) Melhuish, W. H. J. Phys. Chem. 1981, 65, 229. (16) Melhulsh, W. H. J. Opt. SOC.Am. 1962, 5 2 , 1256. (17) Melhulsh, W. H. Rev. Sci. Insfrum. 1982, 33, 1213. (18) Yguerablde, J. Rev. Sci. Instrum. 1988, 3 9 , 1048. (19) Melhulsh, W. H. J. Res. Nati. Bur. Stand., Sect. A 1972, 7 6 , 547. (20) Cehelnik, E. D.; Mielenz, K. D. Appl. Opt. 1978, 15, 2259. (21) Vavilov, S. J. 2.Phys. 1927, 42, 311. (22) t’avllov, S. J. 2.Phys. 1924, 2 2 , 268. (23) Bowen, E. J. Proc. R . SOC.London, Ser. A 1938, 154, 349. (24) Mandal, K. Pearson, T. D. L.; Demas, J. N. Anal. Chem. 1980, 5 2 , 2184. (25) Mandai, K.; Pearson, T. D. L.; Demas, J. N. Inorg. Chem. 1981, 2 0 , 786. (28) Demas, J. N.; Taylor, D. G. Inorg. Chem. 1979, 18, 3177. (27) Buell, S.; Demas, J. N. J. Phys. Chem. 1983, 8 7 , 4675. (28) Mandal, K.i Pearson, T. D. L.; Krug, W. P.; Demas, J. N. J. Am. Chem. SOC. 1983, 105, 701.
.
RECEIVED for review June 26,1984. Accepted September 26, 1984. We gratefully acknowledge support by the Air Force Office of Scientific Research (Grant No. 78-3590) and the NSF (Grant No. 82-06279). The computer system, which is part of the University of Virginia laser facility, was purchased in part with funds ffom NSF Grant 77-09296.
Four-Component Determinations Using Phase-Resolved Fluorescence Spectroscopy Frank V. Bright and Linda B. McGown*
Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74078
A method for the determlnation of the lndivlduai components in a quaternary system of anthracene derlvatlves is presented. The method employs phase-resolved fluorescence spectroscopy (PRFS) In conjunction wlth wavelength selection for the simultaneous determinatlon of anthracene, l-chioroanthracene, 2-chloroanthracene, and 9-chioroanthracene In mixtures Containing these four specles. The design and use of a least-squares routine for the calculation of the amplitude and phase d of the cosine curve that describes the phaseresolved fluorescence Jntenslty (PRFI)as a function of detector phase angle (aD)for each of the Individual components are dlscussed. The fitting of PRFI vs. aDfor mixtures is carried out by using a fifth-order polynomidl routine. The results of the least-squares and flfth-order polynomial analyses are combined to generate a series of simultaneous equations whlch are “solved” for the analytical conceritratlons of the fow components in each mlxture by uslng a Gausslan-Newton iteratlve procedure written In Apple BASIC.
minations (3, 4). The sample under study is excited with time-dependent sinusoidally modulated light, E(t ) ,
E(t) = A ( l
0003-2700/85/0357-0055$0 1.50/0
(1)
where mexis the degree of exciting beam modulation (i.e., the ratio of ac amplitude to the dc intensity component), A is the dc intensity component of the exciting beam, and w is the angular modulation frequency (w = 27rf where f is the linear modulation frequency in hertz, commonly in the megahertz range). The resulting wavelength-dependent (A) fluorescence emission F(X,t ) for a single component sample with exponential decay will be equal in frequency (0)and phase-shifted by an angle Q unique to the fluorescing species, appearing as
F(X, t ) = AL(1
+ mexmsin (wt - a))
(11)
where m is the demodulation factor (cos a). For a solution containing i uncorrelated (noninteracting) fluorophores j
F(X, t ) = The w e of phase-resolved fluorescence spectroscopy (PRFS) for multicomponent fluorescence determinations in which selectivity based on fluorescence lifetime is combined with wavelength discrimination is described for a quaternary system of anthracene derivatives. The theory and instrumentation of PRFS have been described in detail elsewhere (I, 2) and are based on the phase-modulation method for fluorescence lifetime deter-
+ mexsin w t )
C Ax[ (1 + mexmisin ( u t - ai)) (111)
i=l
The time-dependent fluorescence emission is observed with phase-resolved detection, in which F(X,t ) is multiplied by a periodic reference function P ( t )that depends on the detector phase angle (QD)
P ( t ) = 0 from 0’ to detector setting aD P ( t ) = 1 from ap~t o (QD 180’)
+
P(t) = 0 from 0 1984 American Chemical Society
(ad+ 180’)
to 360°
(IV)
58
ANALYTICAL CHEMISTRY, VOL. 57, NO. 1, JANUARY 1985 I @ l , h l = f A @ l , h l C A + ‘lCA@l,hl
*@,A1
= iA@n,hlCA
clCA
+T1cA@n,hlC1~~
T 2CA @ l , hl C 2 C A
i2CA@n,hlC2CA
72CA
and is integrated. The result is a time-independent signal proportional to the cosine difference between the detector phase angle and the phase angle of the fluorescing species @[ I
F(X, @D) =
Ax’T?ZexT?Zl COS
(V)
(@D - @i)
1=1
The first three factors can be combined into a single term I
F(X, @D) =
*At
r=l
cos (@D -
(VI)
which is simply the wavelength-dependent phase-resolved “amplitude” of the phase-resolved fluorescence emission function F(h, @D). Applications of PRFS previously described in the literature have primarily involved the selective “nulling” of one component in a two-component mixture, so that both components can be selectively measured (2, 5 ) . In this approach aD is chosen to be 90’ out of phase with one of the components in f go’), so that the two-component systems (Le., aD= component 1 is “nulled” (its phase-resolved fluorescence intensity (PRFI) contribution is equal to zero) and only component 2 is observed. If @D = a2f go”, only component 1 is observed. Recently, the improved accuracy of simultaneous determinations of two fluorescent species with essentially identical emission and excitation spectra using measurements a t two nonnull phase angles relative to the nulling approach was demonstrated (6). The use of multiple, nonnulling detector phase angles has also been applied to the development of a homogeneous fluoroimmunoassay (7), interferent elimination (B), and simultaneous three-component determinations (9). In the work described here, results for the analysis of a four-component system using phase-resolved fluorescence measurements a t three different emission wavelengths in conjunction with a series of equally spaced detector phase angles are presented. This is the first application of the use of overdetermined systems for multicomponent phase-resolved fluorometric determinations, in which curves of PRFI vs. @D are fit by using a least-squares analysis for the standards and fifth-order polynomial regression fits for the mixtures. Anthracene (A), 1-chloroanthracene (ICA), 2-chloroanthracene (2CA), and 9-chloroanthracene (9CA) were chosen as the four components because of their severe spectral overlap and sufficient relative lifetime differences to allow PRFS discrimination. The method is potentially useful for determination of product distributions of anthracene reactions, especially in samples containing low concentrations (
