327
Anal. Chem. 1984, 56,327-331
number R809474 to L. J. Cline Love, it has not been subjected to the Agency’s required peer and administrative review and, therefore, does not necessarily reflect the view of the Agency and no official endorsement should be inferred. This work
was presented, in part, a t the 23rd Colloquium Spectroscopicum Internationale in Amsterdam, The Netherlands, Abstract No. 62, and at the Southeastern Regional ACS Meeting, Charlotte, NC, in Nov 1983, Abstract No. 161.
Micelle-Stabilized Room-Temperature Phosphorescence with Synchronous Scanning R. A. Femia and L. J. Cline Love*
Department of Chemistry, Seton Hall University, South Orange, New Jersey 07079
The experimental requlrements for synchronous wavelength scannlng mlcelle-stabilized room-temperature phosphdrescence and the factors affecting peak resolution are presented and compared wlth those for synchronous wavelength scannlng fluorescence. Identification of lndlvldual compounds In a four-component mlxture Is Illustrated, and crlterla to ldentlfy and mlnlmlze triplet state energy transfer are glven. Conslderable Improvement In resolution of the synchronous peaks Is obtained via second derlvatlve spectra.
Micelle-stabilized room-temperature phosphorescence (MS-RTP) has been observed for a wide variety of lumiphors ( I , 2) and offers improved selectivity for the analysis of many mixtures, This selectivity results from the fact that not all molecules phosphoresce, thus eliminating certain spectral interferences observed in fluorescence, and that the phosphorescence emission is shifted into the generally less crowded red region of the spectrum. The technique of synchronous wavelength scanning luminescence originally described by Lloyd (3) affords an additional degree of selectivity in phosphorescence and has been employed by a number of workers (4-8). Its use in solid substrate room-temperature phosphorescence has been documented by Vo-Dinh and coworkers (61, where the method was found to work well for compound&meeting certain energy requirements. This paper describes the application of synchronous wavelength scanning to MS-RTP and details the requirements for succesful analysis of multicomponent mixtures by synchronous phosphorescence. A new type of interference probably unique to room temperature phosphorescence in fluid solution, namely, triplet-state energy transfer between components in a sample mixture, is discussed. Factors contributing to resolution of synchronous peaks in both fluorescence and phosphorescence are compared, and a method to improve the resolution wing second derivative spectra manipulation is illustrated.
EXPERIMENTAL SECTION Reagents. All polynuclear aromatic hydrocarbons, except
biphenyl, were obtained from CHEM Service, Inc. (West Chester, PA), as kit # PNA-45 and were used as received. Biphenyl was supplied by MCB (Norwood, OH) and was recrystallized twice from ethanol. Original stock solutions of each analyte were dissolved in spectrograde methanol (Fisher, Springfield, NJ). Electrophoresis grade sodium dodecyl sulfate was obtained from Biorad (Richmond, CA) and was treated with Darco decolorizing charcoal, filtered through a Rainin 0.45-wm pore diameter nylon filter, and then recrystallized twice from methanol. Thallous
nitrate (Fisher, Springfield, NJ) was used as received. Water was deionized and then triply distilled over NaOH pellets. Gases were obtained from AGL Welding (Clifton, NJ). Ultrahigh-purity nitrogen gas was passed through an Alltech Oxy-Trap before entering the sample cuvette. Apparatus. Luminescence spectra were obtained with a SPEX Fluorolog 2+2 spectrofluorometer (SPEX Industries, Metuchen, NJ). The instrument is equipped with double excitation and emission monochromators, with a spectral band-pass of 1.8 nm/mm. The photomultiplier tube (PMT) used was a Hammamatsu R928. Data were acquired in the photon counting mode by a SPEX Datamate computer and stored on floppy disks. Datamate ROM hardwire routines are used for manipulation of the stored spectra via the method of Savitsky and Golay which used a running average method of spectra smoothing (9, 10). The smoothed data are subjected to a linear least-squares regression using other hardwire routines in the Datamate. The regression converts the digitally stored spectra into a continuous function which is then derivatized via a quadratic cubic fit function (10). Hard copies of spectra were obtained through the use of a Houston Instruments digital x-y recorder. The experimental procedure for micelle-stabilizedroom-temperature phosphorescence (MSRTP) have been well documented (I, 2). Optimization of offsets for synchronous luminescence has been described elsewhere (4-6, 8).
GENERAL CONSIDERATIONS Synchronous wavelength scanning luminescence as a means of monitoring the presence of specific lumiphors has been demonstrated by Baudot and Andre (11). Synchronous wavelength scanning micelle-stabilized room-temperature phosphorescence is carred out in a similar manner by scanning the excitation and emission monochromators a t a fixed rate with a constant wavelength offset maintained between them. The value of the monochromator offset is given by the wavelength difference between the longest wavelength peak in the excitation spectrum and the shortest wavelength peak in the phosphorescence emission profile. The synchronous scanning MS-RTP spectrum of fluorene is shown in Figure 1. For this molecule, the longest wavelength excitation peak is also the excitation wavelength maximum at 303 nm, and this is convolved with the phosphorescence emission peak at 439 nm through the use of a 136 nm offset to obtain the synchronous peak. The instrument used for the synchronous wavelength scanning mode plots the emission intensity vs. wavelength position of the excitation monochromator. Thus, a t any given point in the synchronous scan, the position of the emission monochromator is given by A, = A,, + Ah, AA being the wavelength offset for any given scan. Thus, the emission wavelength axis should have the wavelength offset value added to it to obtain the actual emission wavelength. Fluorene, which has an excitation maximum a t 303 nm and
0003-2700/S4/0356-0327$0 1.50/0 0 1984 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 56,NO. 3, MARCH 1984
328
1
+
n '
L
= +
1
I
VI
C (u
t
i i
+,
C
H
i I
\ I
J
i
r
200.00
400. 00
Xavel e n g t h
6C0.9:
+
an emission maximum a t 439 nm, produces a synchronous peak at 303 nm when run with the optimum wavelength offset of 136 nm. Effect of Slit Width. For a large number of fluorescent molecules, the optimum wavelength offsets are small, with values of < l o nm not uncommon. This imposes contraints on the slit width for optimum resolution and signal-to-noise ( S I N )ratio. Generally, for synchronous luminescence, the spectral band-pass of the fluorometer must be less than the wavelength offset to prevent transmission of a portion of the excitation light to the PMT. The requirement of narrow slit widths for synchronous wavelength scanning fluorescence for many molecules diminishes the intensity of the exciting radiation and the subsequent synchronous fluorescence emission intensity. This decrease in signal is further compounded if the excitation wavelength is located in the proximity of the emission wavelength such that stray or scattered light reaches the PMT introducing a background noise component. These problems are avoided in synchronous wavelength scanning micelle-stabilized room-temperature phosphorescence by virtue of the considerably larger wavelength offsets involved, thus allowing the use of wide slit widths for maximum excitation and emission efficiency.
Resolution in Synchronous Wavelength Scanning Luminescence. Lloyd and Evett have described the factors affecting resolution and peak intensity for synchronous wavelength scanning fluorescence (4, 6). Generally, synchronous fluorescence will exhibit better resolution (smaller peak half-width) than synchronous phosphorescence. This can be easily rationalized by examining the spectral features of the respective spectra. Fluorescence spectral profiles for many compounds exhibit a mirror image symmetry relationship with peaks in the excitation spectrum and have more fine structure compared to phosphorescence spectra. For a compound which displays good symmetry between its excitation and emission profiles, the resulting synchronous peak will have good resolution as a result of the fact that the 0bands used to determine the fluorescence offset will rise and fall simultaneously. However, in MS-RTP there is usually very little symmetry between the excitation and phosphorescence emission profiles, and phosphorescent compounds generally do not exhibit sharp, vibronic transitions at room temperature, as do fluorescence spectra a t room temperature. Therefore, when the wavelength offset between the longest wavelength excitation band and the shortest wavelength phosphorescence band is used, the intensities of the two peaks will not necessarily rise and fall in sequence, depending on the vibronic fine structure of the phosphorescence. If the shortest wave-
:
200. 00
In m l
Synchronous MS-RTP peak obtained for a 1.2 X lo-' M fluorene solution with a wavelength offset of 136 nm: slits, 8 mm EX and EM; peak maximum at 303 nm excitation wavelength; actual emission wavelength = 303 136 = 439 nm. Figure 1.
t
1
I
Li,
47s. 08
I
750.~0
Wavel e n g t h [ nrn) Flgure 2. Total luminescence profile for benzo[elpyrene: excitation wavelength = 332 nm; slits, 8 mm EX, 2 mm EM for luminescence, 2 mm EX, 8 mm EM for excitation profile.
/\
1 7, + .-
I
+
i-
u C
i 1
200.00
/
/i
I,--
\
c-_
400. 00
I
t
600.00
Wavel e n g t h [ nml Flgure 3. Comparison of synchronous fluorescence and synchronous MS-RTP peaks for 1.8 X lo-' M benzo[e]pyrene: wavelength offsets, fluorescence = 63 nm, MS-RTP = 223 nm; peak maximums at 332 nm excitation wavelength; actual emission wavelengths = 332 nm 4offset.
length phosphorescence band is close to the phosphorescence intensity maximum, a significant portion of the phosphorescence will be transmitted to the PMT as the excitation and emission monochromators pass the peak maxima used to determine the wavelength offset, thus contributing to overall band broadening of the observed synchronous peak. These effects are illustrated in Figure 2, which shows the total luminescence profile of benzo[e]pyrene and the relationships between excitation and fluorescence spectra, and excitation and phosphorescence spectra used to select a wavelength offset value. Figure 3 compares the synchronous fuorescence and synchronous MS-RTP signals for benzo[e] pyrene, the former showing considerably better resolution. In contrast to conventional fixed excitation wavelength luminescence, slit width does not have as much influence on spectral resolution in synchronous phosphorescence. In synchronous MS-RTP, resolution is more a function of the inherent resolution of the vibronic transitions in the phosphorescence profile, which is low in the fluid, room-temperature, nonrigid matrix employed. Consequently, slit widths used in synchronous MS-RTP should be large to achieve maximum intensity and sensitivity without any appreciable loss in resolution. Second-Order Radiation Interference. A frequent spectral interference of grating second-order radiation transmission is encountered in synchronous MS-RTP which employs large wavelength offsets. For example, if the syn-
ANALYTICAL CHEMISTRY, VOL. 56, NO. 3, MARCH 1984
Table 1. Synchronous Micelle-Stabilized Room-Temperature Phosphorescence Excitation Wavelengths and Optimum Wavelength Offsets for Selected Polynuclear Aromatic Hydrocarbons excitation wavelength, wavelength compound nm offset, nm biphenyl phenanthrene triphenylene fluorene fluoranthene chrysene pyrene benzo[a]pyrene benzo[ elpyrene dibenzofuran dibenzothiophene
27 2 298 287 303 362 305 335 388 332 300 315
180 185 152 136 243 21 5 268 311 223 131 105
chronous wavelength MS-RTP scan of pyrene is run from 200 to 600 nm with a wavelength offset of 268 nm, a second-order spectral interference is seen when the emission monochromator reaches 536 nm, a wavelength corresponding to two times the excitation wavelength, while the synchronous pyrene peak will occur at 335 nm. Higher order radiation interferences will always occur at multiples of the wavelength offset if the excitation monochromator is scanned over the wavelength numerically equal to the wavelength offset. The most obvious way to avoid second-order interference peaks is to change the wavelength interval over which the fluorometer is scanned. In the case of pyrene, initiating the synchronous wavelength phosphorescence scan at 300 nm will effectively remove the peak. A second method of removing second-order spectral interferences is the use a background subtraction approach unique to MS-RTP. One of the requirements for inducing MS-RTP is that the sample must be deaerated to remove oxygen, a triplet state quencher (12). To background subtract, a synchronous spectrum containing fluorescence, phosphorescence, scatter, and second order peaks is obtained on a deaerated sample. A second synchronous spectrum of an aerated sample is obtained which contains all of the spectral components except the phosphorescence. By simply subtracting the two spectra, a synchronous spectrum of only the phosphorescence is obtained. This approach is subject to some over- or undercorrection and should be used with care (13). Any second-order spectral interferences observed in the spectra presented here were removed by using the background subtraction method.
RESULTS AND DISCUSSION Selectivity. In fluorescence, many structurally dissimilar compounds have very similar Stokes shifts (5)and very similar wavelength offsets in synchronous fluorescence. Thus, in the analysis of mixtures a compromise wavelength offset is chosen to give the largest number of resolved components in a single scan. For a mixture of lumiphors with fluorescence profiles that are spectrally well separated from one another, this approach is useful in identifying fluorescent species. However, for mixtures of fluorescent compounds which are not spectrally resolved, vibronic conjestion in the wavelength region of interest will result in poorly resolved synchronous peaks. On the other hand, phosphorescent compounds which differ in structure by the number or placement of aromatic rings have MS-RTP wavelength offsets which are unique to each family. Table I gives a listing of selected PNAs and their optimum phosphorescence wavelength offsets. As shown, the offsets vary widely and are, for the most part, quite different from each other. This lends synchronous MS-RTP an additional degree of selectivity over synchronous fluorescence. A mixture
n I\
4
I
T
i1
680. 00
40d.00
200.00
329
Wavel e n g t h ( n m ) Figure 4. Synchronous MS-RTP spectra of four-component mixtures described in Table 11: (0)peak at 303 nm from fluorene using 136 nm offset, and (-) peak at 335 nm from pyrene using 268 nm offset; actual emission wavelengths = excitation wavelength offset.
+
t
I
t
I
I
1
t I
208.00
J :
I 4130.00
Wavel e n g t h
600.03
(nml
Figure 5. Synchronous MS-RTP spectra of four-component mixture described in Table 11: (0)peak at 287 nm from triphenylene using 152 nm offset, and (-) peak at 298 nm from phenanthrene using 185 nm offset; actual emission wavelength = excitation wavelength offset.
+
Table 11. Concentrations and Optimum Synchronous MS-RTP Wavelength Offsets for Four-Component Mixture of PNAs Used to Obtain Spectra in Figures 4 and 5 wavelength compound concn, M offset, nm pyrene fluorene triphenylene phenanthrene
2.5
X
7.7 x
7.68 X 7.18 X
26 8 136 152 185
can be scanned at a specific offset which is entirely unique to one compound or family of compounds in the mixture. By use of different phosphorescence offsets, it is possible to selectively identify compounds in a mixture based on differences in their So TI spacings. This can be analytically useful for monitoring the presence of a specific phosphorescent species in the presence of a wide variety of other emitting impurities, such as in environmental monitoring of specific toxic substances. The selectivity in synchronous MS-RTP is illustated in Figure 4 and 5 for the synchronous MS-RTP spectra of a mixture of pyrene, fluorene, triphenylene, and phenanthrene. Table I1 lists the concentrations of each component and their respective wavelength offsets. Four successive synchronous wavelength scans were run at each of the four offsets in order
-
;I
330
ANALYTICAL CHEMISTRY, VOL. 56, NO. 3, MARCH
1984
t
s
(u
-P C H
208.88
Navel e n g t h
(nml
c
4118.88
Wave) e n g t h
683.83
(nml
Figure 6. Emission spectra of two mixtures of 1.8 X lo-' M phenanthrene dissolved in 5.6 X lo-' M biphenyl (dotted line) and in 1.1 x M biphenyl (solid line): fixed excitation wavelength = 272 nm, Optimum for biphenyl: slits, 8 mm EX, 2 mm EM.
Figure 7. Second derivative spectrum of synchronous MS-RTP spectrum run with 152 nm offset shown in Figure 6: peak at 287 nm from triphenylene, peak at 300 nm from phenanthrene: compare resolution obtained with that in Figure 5.
to optimize selectivity in component identification. Figure 4 shows the synchronous phosphorescence peaks obtained for the four-component mixture when scanned a t the optimum offsets for fluorene and pyrene. The fluorene peak occurs at 303 nm with a wavelength offset of 136 nm, and the pyrene peak occurs a t 335 nm with a wavelength offset the 268 nm. Figure 5 contains the synchronous scans of the four-component mixture run at the optimum offsets for triphenylene and phenanthrene. The triphenylene peak at 287 nm was obtained with a wavelength offset of 152 nm, and the phenanthrene peak occurred a t 298 nm using a 185-nm wavelength offset. The synchronous wavelength scan of a solution containing only triphenylene was much better resolved than that shown in Figure 5. The band broadening observed for triphenylene in the mixture is believed to be due to the presence of phenanthrene whose vibronic bands overlap those of triphenylene. Similarly, phenanthrene also exhibits slight band broadening of its synchronous peak on the short wavelength side, due to vibronic interference from triphenylene. Energy Transfer Interferences. The occurrence of triplet state energy transfer in fluid solution is a well-documented phenomena in luminescence spectrometry (14,15). This type of interference can occur in the analysis of mixtures by synchronous wavelength scanning MS-RTP, causing distortions in the spectra and inaccurate analysis. Mention of triplet state energy transfer interference has not been recorded in the literature to date on solid-substrate room-temperature phosphorescence. T o occur in that regime, the interacting species would have to be initially deposited physically near one another. Triplet state energy transfer in synchronous MS-RTP is illustrated for the determination of biphenyl and phenanthrene. The excitation maxima and optimum offset values for biphenyl and phenanthrene are 272 nm and A180 nm, and 300 nm and A185 nm, respectively. Note that these compounds have similar wavelength offsets and their total luminescence profiles are separated by 28 nm, making them good candidates for resolution by a single synchronous scan run at an offset of 183 nm. Figure 6 gives two emission spectra of two different solutions of biphenyl and phenanthrene. In each case, the phenanthrene concentration was held constant a t 1.8 X lo6 M, while the biphenyl concentration was varied. Both solutions were excited at 272 nm, the optimum excitation wavelength for biphenyl. The solid line represents a biphenyl concentration of 1.1X M and the dotted line corresponds to a biphenyl concentration of 5.6 x IO4 M. The triplet state emission profile obtained in both cases is identical with the spectrum of phenanthrene (not shown). The higher concentration of biphenyl sensitizes the phenanthrene emission, most
probably through triplet-triplet energy transfer. The triplet emission of biphenyl does not overlap the excitation spectrum of phenanthrene, thus eliminating direct triplet-singlet energy transfer. Energy transfer can cause significant errors in both qualitative and quantitative characterization of mixtures. To experimentally determine if energy transfer is occurring in a mixture of unknown lumiphors, the solution should be studied a t varying dilutions. As the triplet emitter concentration decreases, the rate of phosphorescence emission will become competitive with the rates of diffusive encounters and energy transfer between triplet-triplet-donor acceptor pairs. At lower concentration, new triplet emissions will appear from species which act as donors at higher concentrations. Since this phenomena in fluid solution is a diffusion limited process (16,17),it is desirable to work at low concentrations of analytes to minimize this effect. Data acquired in our laboratory, as well as in many others, indicate that optimum results are obtained with solutions having a total analyte concentration of
