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ANALYTICAL CHEMISTRY, VOL. 50, NO. 14, DECEMBER 1978
when 2,6-pyridine-dicarboxylicacid was subjected to the reagent conditions. Thus it appears that some p-hydroxypyridine could be formed. If so, it should nct be possible to hydroxylate a 2,4,6-trisubstituted pyridine. The fluorescence spectrum of 2,4,6-trimethylpyridine remained the same after it was treated with the reagents. Hence, if 0 - and p - isomers are the products, the yield would be 54% or greater. Analysis by Hydroxylation. The analytical method is applicable to several pyridine compounds tested in the range 104-10-4 M. Table I1 shows the gain in analytical sensitivity after hydroxylation of the various pyridine compounds. Gain is defined by the following expression: F(sample after hydroxylation)- F(reagent) F(samp1e
in
water) - Ftwater)
where F(sample after h&,.&tlon) = fluorescence intensity of sample after hydroxylation, F(reagent) = fluorescence intensity of reagent blank treated under the same procedure, F(jample In water) fluorescence intensity of aqueous solution of sample, and F(uaterj = fluorescence intensity of water. There is no p H dependence on F(sample ,=water) for the samples listed in Table 11. Calibration plots were linear.
Some heterocyclic compounds do not yield an analytical gain when subjected to t h e hydroxylation method. Thus quinoline fluoresces strongly in acidic medium, whereas after hydroxylation a quinoline solution fluoresces (but less intensely) in acidic and alkaline media. T h e effect of hydroxylation is, in this case, to decrease analytical sensitivity, though the altered p H dependence of the fluorescence may prove to be analytically useful.
LITERATURE CITED (1) G. A. Hamilton and J. P. Friedman, J. Am. Cbem. Soc., 85, 1008 (1963). (2) G. A. Hamilton, J. P. Friedman, and P. M. Campbell, J . Am. Cbem. SOC., 88, 5266 (1966). (3) G. A. Hamilton, J. W. Hanifin, and J. P. Friedman, J . Am. Cbem. Soc., 88, 5269 (1966). (4) K . A. Connors and K . S. Albert, Anal. Cbem., 44, 879 (1972). (5) K. S. Albert and K. A. Connors, J . Pharm. Sci., 62, 625 (1973). (6) C. J. Eguchi, Bull. Cbem. SOC.Jpn., 2, 180 (1927). (7) R. C. Weast, "Handbook of Chemistry and Physics", 53rd ed., Chemical Rubber Co., Cleveland, Ohio, 1972. (8) H. D. Dakin, Org. Syn., 1, 143 (1932). (9) K. S. Albert, P h D dissertation, University of Wisconsin, Madison, Wis., 1972 (10) A. Albert and A. Hampton, J , Chem. Soc., 507 (1954)
RECEIVED for review May 30, 1978. Accepted September 1, 1978.
Singlet-Triplet Energy Difference as a Parameter of Selectivity in Synchronous Phosphorimetry T. Vo-Dinh" and R. B. Gammage Health and Safety Research Division, Oak Ridge National Laborafory, Oak Ridge, Tennessee 37830
The direct use of the singlet-triplet energy difference as a new factor of selectivity in phosphorimetry is suggested. The methodology, which is based on the synchronous excitation technique, exploits the specificity of energy gaps AsT between the phosphorescence emission band (T, So) and the So or S, So). This approach can absorption bands (S, greatly improve the selectivity in phosphorescence analysis. Some aspects of the specificity of the parameter AA in the characterization of polynuclear aromatic (PNA) compounds are discussed. The differentiation of isomeric PNAs is illustrated and the qualitative analysis of a Synthoil sample is given.
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T h e performance of a n analytical method in multicomponent analysis is often characterized by the variety of selectivity factors it offers. Regarding this aspect, luminescence spectrometry offers the choice of selecting independently two wavelength variables (excitation and emission). The specificity of luminescence spectrometry can be enhanced by using special techniques to exploit these spectral variables. A modulation technique based upon a slight difference in spectral bands for selectively obtaining excitation and emission spectra of a specific component in a mixture of two fluorophores has been reported ( I ) . Other features (2, 3) of the spectral structure have also been amply exploited by using quasi-linear spectroscopy a t low temperatures, especially with Shpolskii solvents ( 3 ) and other matrices ( 4 ) . Alternatively, time-resolved and phase-resolved spectroscopies (5,6),based on the differences in the lifetime of the excited states have 0003-2700/78/0350-2054$01.00/0
opened u p new analytical possibilities. We first report here the exploitation of a physical parameter of the excited states, namely the singlet-triplet energy difference (AsT), as a direct factor of selectivity in phosphorimetric analysis. Although AST is a well-known physical concept, to the authors' knowledge it has never been exploited explicitly as a factor of selectivity. In the past, t h e energy levels of the excited singlet and triplet states of organic molecules have been used in conventional spectroscopy to characterize them, but the difference in energy between t h e excited singlets Sl(or S,) and the lowest triplet TI (from which the compound phosphoresces) has not yet been used experimentally to identify compounds in multicomponent analysis. The use of this singlet-triplet energy gap employs the so-called synchronous luminescence technique where the excitation and emission wavelengths are scanned simultaneously (7). In an earlier publication, a general methodology for this technique in fluorimetry was reported (8); it was demonstrated that this synchronous technique is not limited merely to providing finger-print signatures of complex samples such as oil spills ( 9 ) ,but it also can provide more specific information about t h e molecular size and type of the components. A similar approach applied to phosphorescence analysis is reported in this study. Although the experimental procedures for synchronous scanning in phosphorimetry are the same as those described previously in fluorescence analysis ( 8 ) ,the photophysical principles involved in the two types of applications are fundamentally different. The procedure t o "sort out" selectively a specific emission signal of one component in a mixture is described. An analysis of several C 1978 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 50, NO. 14, DECEMBER 1978
polynuclear aromatic (PNA) compounds of environmental interest will illustrate the usefulness of the method. Finally, a brief discussion about the analytical suitability of the parameter AST in the analysis of P N A compounds is given. In this work, the synchronous method has been applied to Room Temperature Phosphorimetry (RTP). This relatively new analytical technique is based on phosphorescence a t room temperature from organic compounds adsorbed on filter paper (10-14). T h e R T P method, as opposed to traditional lowtemperature phosphorimetry, does not require the use of cryogenic equipment and has a great potential for organic pollutant monitoring. The differentiation of isomeric PAH (1,2,5,6-dibenzanthracene and 1,2,3,4-dibenzanthracene) by synchronous phosphorescence analysis is illustrated. T h e qualitative identification of pyrene in a Synthoil extract is shown.
EXPERIMENTAL Apparatus and Procedure. All phosphoroscopic measurements were performed with a Perkin-Elmer spectrofluorimeter (Model 43A, Perkin-Elmer, Norwalk, Conn.) equipped with a rotating cylinder. The excitation light source was a 150-W xenon arc lamp. The detector was an R777 photomultiplier (Hamamatsu Company, Middlesex, N.J.) with a spectral response from 185 to 850 nm. Compared with the R508 photomultiplier used in earlier work (a),this photomultiplier offers a better spectral response in the long wavelength region (>E100nm) where phosphorescence of most high-ring-number PNA compounds occurs, enabling one to improve the sensitivity by at least one order of magnitude. A spectral bandpass of 5 nm was used for the excitation and emission monochromators. For synchronous luminescence measurements, both excitation and emission monochromators were locked together and scanned simultaneously using a slow scan rate of 0.25 nm s-’. No correction for instrumental response was made. A laboratory-constructed sample holder was similar to the one described in previous work (13). Details of the sample preparation for RTP measurements were reported previously (11-14). The detection limits of most PNA compounds that have been studied were in the sub-nanogram range. Reagents. The PNA compounds investigated in this work were purchased and used without further purification: acridine, benzo[a]pyrene, benzo[a]pyrene, 1,2,3,4- and 1.2,5,6-dibenzanthracene, chrysene, fluorene, pyrene (Aldrich Chemical Co., Milwaukee, Wis.), and naphthalene (Fisher Scientific Co., Fair Lawn, N.J.), quinoline and phenanthrene (Matheson Coleman and Bell, Norwood, Ohio). A mixture of ethanol and water (3:1, v/v) was used as solvent. The substrate was filter paper (491-c type) purchased from Schleicher and Schuell, (Keene, N.H.). The Synthoil sample was obtained from the Analytical Chemistry Division of the Oak Ridge National Laboratory. The extractions for the acidic, basic, and neutral fractions were carried out essentially as described by Swain et al. ( 1 5 ) . The final ethyl ether portion was used for RTP analysis. Lead acetate was used as heavy atom perturber in the phosphorimetric measurement of the dibenzanthracene mixture and the Synthoil sample. RESULTS AND DISCUSSION Application of the Synchronous Excitation Method to Phosphorimetry. A conventional phosphorescence emission spectrum EMp(A)can be obtained by scanning the emission monochromator wavelength A while the compound is excited a t a fixed excitation wavelength A‘. On the other hand, a phosphorescence excitation spectrum EXP(A’)is obtained by varying t h e excitation wavelength A‘ while t h e phosphorescence emission is observed a t a fixed wavelength. A third possibility is to scan simultaneously both A and A‘ a t a constant interval AA = (A - A’). I t was shown (8) that the synchronous signal Zsp is given by the relation Isp(A, A’) = h.c.Exp ( A’) .EM?,( A) (1) where h = a n experimental constant factor, c = the concentration of the analyte, Zsp = synchronous phosphorescence signal, E X P= excitation phosphorescence spectrum, EMP=
2055
h
AST
Figure 1. Influence of phorescence signal
Ah
and AsT on the synchronous phos-
emission phosphorescence spectrum, A = emission monochromator wavelength position, and ,\’ = excitation monochromator wavelength position. Equation 1 shows that the synchronous signal is a function of both excitation monochromator and emission monochromator wavelengths. In order to illustrate the effect of t h e parameter Ai, let us consider in detail the expression for synchronous phosphorescence. If we set A’ = A - AA, Equation 1 can be expressed as:
Isp(X) = h*c’Exp(X - AX)*EM?p(X)
(2)
Although the form of Equation 2, with EXpexpressed as a function of t h e emission wavelength obscures its physical meaning as an excitation spectrum, this formulation is nevertheless useful in revealing t h e advantages of t h e synchronous method in phosphorimetry. As expressed in Equation 2 and schematically illustrated in Figure 1, t h e synchronous phosphorescence signal Isp is, mathematically, the result of the product, or “multiplicative mapping” of the function EMPand the function EXPthat has been translated by AA on the wavelength scale. From this conceptualization, it can be easily shown that the synchronous signal depends directly upon the overlap of Exp(A- AX) and EMp(A),Le., the value of AA and AsT. In Figure I, AsT shows the energy gap between the first emission band (from t h e left) of t h e phosphorescence spectrum and the first band (from the right) of the excitation spectrum. If the phosphorescence emission and the absorption spectra have intense 0-0 bands, AST represents the energy gap between the first excited triplet T1 and the first excited singlet S1 (also called “singlet-triplet splitting”). B u t if S1 is a weak or insignificant absorption band, one can consider higher excited singlet states (S2,S3, or S,)that show strong absorption. In those cases, AST should be taken as the energy difference between S, and T1. This consideration is important for compounds such as polynuclear aromatic hydrocarbon (PAH) systems or compounds t h a t usually show a weak S l ( a band) system ( t 102-103),whereas their higher excited state S2(p or /? bands) has a stronger oscillator strength ( t 104-105) ( 1 5 ) . If t h e experimentally selected wavelength interval AAl, is larger than the singlettriplet splitting, a synchronous phosphorescence peak will be observed (Figure Ib). On the other hand, if another AA(=AA2) is experimentally chosen to be smaller than AST, the synchronous phosphorescence signal of t h e corresponding compound cannot be observed since there is no overlap between the two functions EXP(A - AX2) and E,(A) (Figure l ~ ) . I t is therefore possible to exploit AST as a direct factor of selectivity; in a multicomponent mixture, it is possible to
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 14, DECEMBER 1978
\
\$ 400
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$ 8 '
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I
1
400
1
I
I
I
I
500 WAVELENGTH
I
I
1
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I
I
I
I
600
I
nm )
Figure 2. RTP spectra of fluorene (50 ng). (a) Conventional spectrum (AX = 125 nm)
(Aex = 279 nrn). (b) Synchronous spectrum
discriminate against components having various AST by a proper choice of AA values. Essentially, the synchronous scan concept with a specific wavelength interval between the excitation and emission takes advantage of a "moving mask'' which can directly exploit the singlet-triplet energy differences. Synchronous RTP Spectrum of Polynuclear Aromatic Hydrocarbons. An example of a practical application of the methodology suggested above is the analysis of organic compounds by the R T P technique. The detection limits by R T P for a large variety of PNA compounds are low (subnanogram range) (14). We are therefore interested in improving this simple technique which has a potential for monitoring organic trace pollutants. One limitation of this method arises from interferences due to the frequent overlap of other phosphorescent species in the sample. Although it is possible that both excitation and emission wavelengths may be varied to minimize interferences. in practice, the usually broad and structureless nature of the RTP spectra often renders these two degrees of freedom ineffectual. Figure 2a shows the conventional R T P spectrum of 50 ng of fluorene using a fixed excitation wavelength of 279 nm. The spectrum exhibits two resolved bands centered a t 430 nm and 458 nm. The broad band located a t ca. 483 nm originates, at least partly, from the background phosphorescence emission of the paper substrate. The synchronous phosphorescence signal of the same fluorene sample using LA = 125 nm is shown in Figure 2b. The AA value was used in order to match the two 0-0 bands (SI Soat 305 nm and T, So a t 430 nm) in the excitation and phosphorescence spectra. Under these conditions, the synchronous signal is characterized by only one sharp peak a t 430 nm. The less intense and broad wing on the long wavelength side of the emission peak corresponds to the paper substrate signal. Whereas the fixed excitation R T P spectrum of fluorene covers a large spectral range from 420 nm to 620 nm, the synchronous signal shows simply one band having approximately a IO-nm spectral width. Clearly,
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Figure 3. RTP spectra of a mixture of fluorene (20 ng), phenanthrene (50 ng), and pyrene (80 ng). (a) Conventional fixed excitation spectrum (Aex = 300 nm). (b) Synchronous spectrum of the same sample (AA
=
125 nrn)
the ability for such a spectral reduction in the phosphorescence emission is highly desirable. The simultaneous determination of multiple phosphorescent components will, consequently, encounter fewer interferences and spectral overlaps. Multicomponent Analysis. A significant advantage of using the AsT selection approach for multicomponent phosphorescence analysis is illustrated in Figure 3. To demonstrate the analytical usefulness of this method, a sample containing 20 ng of fluorene in the presence of 80 ng of pyrene and 50 ng of phenanthrene was analyzed. With a fixed excitation wavelength a t 300 nm, the R T P spectrum of the mixture consists of broad spectral bands overlapping one another (Figure 3a). The band a t 430 nm corresponds to fluorene whereas the peak a t 508 nm is mainly associated with phenanthrene. Pyrene gives an emission which appears as a weak band a t 600 nm. Spectral overlap between these compounds and the paper emission contribute to a diffuse background emission with unresolved broad bands (Figure 3a). Although fluorene emission can be detected as a weak shoulder a t 430 nm, spectral interference from the emission of the two other compounds, as discussed previously, hinders the quantitative analysis. The peak a t 470 nm results from the overlap of two emission bands from both fluorene and Phenanthrene. Note that the RTP spectrum of phenanthrene occurs between 440 to 620 nm. This means that the analysis of fluorene that phosphoresces a t a similar spectral range, could be subject to severe interferences from phenanthrene. The improved selectivity from using the singlet-triplet energy differences in the synchronous method is illustrated in Figure 3b. This figure shows the synchronous phosphorescence spectrum of the same sample using LA = 125 nm. This wavelength interval L A , as discussed in Figure 2, corresponds to the optimal value for fluorene. The wavelength intervals AA that are appropriated for phenanthrene must be I70 nm (difference between the S2 Soband a t 290 nm and the TI Sophosphorescence band a t about 460 nm) or 210 nm [difference between the S3 Soband at 250 (nm and the T1 So band a t 460 nm). Note that since the first absorption band of phenanthrene S1 So('L~)is very weak ( t 2501, a smaller value AA of 130 nm chosen to match SIand TI would give rise to a nonsignificant synchronous signal. These results are reported in Table I. Similar arguments indicate that a good AA value for pyrene is 250 nm ulhich matches TI (at 600 nm) and S2 (at 350 nm). This shows that a AA value of 125 nm chosen for fluorene is smaller than those that are suitable for phenanthrene and pyrene. This important feature can therefore be used to exploit the method discussed previously.
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Table I. Singlet-Triplet Properties of Phenanthrene, Pyrene, and Fluorene
compounds
triplet data, phosphorescencea emission peak, nm
phenanthrene
460
pyrene
600
fluorene
singlet data phosphorescence assignmentsb of corresponding excitation bands, absorption band nm 330 390 250 360 350
(W)d (M)e
S I ( €= 250) S 2 ( e = 1 4 800) S3(c = 67 000) S,(e = 5 1 0 ) S2(e = 5 5 000) S,(€ = 54 0 0 0 ) S I ( €= 1 0 0 0 0 ) S2(e = 19 400)
(S)f (W)
(S) 780 ( W ) 305 (S) 270 (W)
430
experimental optimal a h c values, nm
An optical A h value is dea Taken as the first intense phosphorescence emission band. Data taken from Ref. 1 5 . W = weak intensity phosfined as the wavelength difference between the emission peak and a strong excitation peak. S = strong intensity phosphophorescence excitation band. e M = medium intensity phosphorescence excitation band. rescence excitation band. 7 SYNCHRONOUS RTP SPECTRA OF A MIXTURE 0 DBA ISOMERS
SYNTHOIL SAMPLE SPOTTED ON PAPER
- WITH A X = 2 5 2 nrn --_ WITH A X = 2 7 4 nrn
6
--
2,
.-
- SYNTHO I L
v) u c z 4
---
--P
2 5
PURE PYRENE
( A X = 250 nrn)
0)
c
u)
0
.-
.-"
- 3 J
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0
l
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l
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l
Figure 5. Identification of pyrene in Synthoil by synchronous R T P analysis (U= 250 nm): spectrum of Synthoil (solid curve):spectrum of pure pyrene (dashed curve)
700
Figure 4. Synchronous R T P spectra of a sample containing 4.2 ng of 1,2,5,6-DBAand 42 ng of 1,2,3,4-DBA(see text)
Synchronously scanning the phosphorescence spectrum with AA = 125 nm yields mainly the emission of fluorene, the signals of t h e other two components being practically "nulled out." Figure 3b shows only t h e synchronous phosphorescence spectrum of fluorene free from spectral interferences from the two other compounds. T h e quantity of fluorene determined in t h e presence of pyrene and phenanthrene was 20 ng. T h e standard deviation of the RTP measurements was less than 5%. This observation portends significant advantages for the use of synchronous techniques in phosphorimetry. The unique aspect of this singlet-triplet selection technique is t h a t it allows the signal of a specific component of a mixture to be recorded independently, even though t h e conventional phosphorescence spectra overlap severely. As an illustration of t h e method, we have considered only a simple mixture of three compounds, but the technique becomes particularly advantageous in t h e analysis of more complex samples containing organic compounds of different chemical nature, as discussed in t h e next section. Application to Characterization of PAH Isomers and Analysis of Environmental Sample. Precise identification of P A H isomers is extremely important in environmental
studies and health effect assessment because the toxicity varies considerably with the structure of isomers. Benzo[a]pyrene (BaP) and benzo[e]pyrene are well known examples: whereas BeP is relatively innocuous, B a P is a hazardous compound ( 1 6 ) . Isomeric benzopyrenes can be easily differentiated by exploiting the differences of their singlet-triplet splitting (Table 11). It is noteworthy that phosphorimetry (even with fixed excitation) is a very practical and efficient method for monitoring BaP because its phosphorescence occurs a t the 690-nm range t h a t is not interfered by BeP (543 nm) and by most of t h e other PAHs investigated (Table 11). Another (1,2,5,6-DBA). example of interest is 1,2,5,6-dibenzanthracene This compound is reported to have a much stronger carcinogenic activity than t h e isomer 1,2,3.4-DBA (16). Differentiation between these two isomers when both compounds are present in a mixture is more difficult than with benzopyrenes because their conventional phosphorescence spectra, shifted with respect to each other by only 10 nm, strongly overlap. The use of t h e synchronous technique can greatly facilitate the identification of each individual DBA isomer in mixtures. Figure 4 shows that 1,2,5,6-DBA (4.2 ng) can be easily identified in a binary mixture that contains 10-fold in excess of 1,2,3,4-DBA (42 ng): the wavelength interval used for 1,2,5,6-DBA was 254 nm and for 1,2,3,4-DBA was 274 nm. Figure 5 illustrates a practical application of synchronous phosphorimetry t o the analysis of an mvironmental sample,
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 14, DECEMBER 1978
Table 11. RTP Excitation and Emission Bands and Experimental A h Values for Several Polynuclear Aromatic Compounds experimental excitaoptimal emission Ah tion peaks, values, peaks, nm compounds nm nm acridine chrysene fluorene naphthalene phenanthrene pyrene qu in ol ine benzo[e]pyrene benzo[a]pyrene 1,2,5,6-DBA 1,2,3,4-DBA
360 325 305 275 250 350 315 335 392 301 296
640 51 5 430 472 460 600 46 5 543 690 555 570
280 190 125 197 21 0 250 150 208 298 254 274
an extract of Synthoil. Previous analysis by gas chromatography has identified pyrene as one of the most abundant compounds in this sample of Synthoil(17). Synchronous R T P can offer a rapid and simple method to search for pyrene in this sample without resorting to separation techniques. With AA = 250 nm (Table 11), a sample of pure pyrene exhibits a peak a t 597 nm (Figure 5 , dashed curve). Under the same experimental conditions, the synchronous R T P signal of Synthoil (Figure 5, solid curve) reveals the presence of pyrene by the narrow band a t 595 nm. A detailed description of the characterization of P N A in various synfuel samples by R T P is given elsewhere (18). The example shown here in Figure 5 illustrates the basic separation of the synchronous technique: only the signal from the analyte of interest (with optimal AX value) is enhanced and exhibits narrow-band structure whereas spectral interferences from the other components in the sample are decreased, giving rise to a broad and featureless background. Analytical Specificity of t h e P a r a m e t e r A s p (i) Since the first triplet energy level (T,) is lower than the first excited singlet energy level (Sl),there is always a positive energy gap AsT. I t is therefore possible to "null out" the synchronous phosphorescence emission of a compound by using a AA value smaller than that specific AsT, deleting the "nulled" component spectrum from the emission spectrum. (ii) AST can be used as a selectivity parameter indicating the size of PAH compounds since the singlet-triplet splitting depends upon the ring size (number of the benzenoid rings). An important group of compounds which reflect this basic rule is the family of linear polyacenes (19,20). Similar rules apply for other aromatic compounds, such as the cyclic polyacenes, although the variation of AsT with the ring size is less remarkable. This feature suggests that, in those cases where a mixture of many polyacenes of various sizes has to be analyzed, it would be possible to use different AX values in order to analyze selectively a specific compound or group of compounds. In order to "null out" the emission of compounds of a certain ring size, the experimentalist must use a wavelength interval AA smaller than the singlet-triplet splitting t h a t corresponds to this class of compounds. (iii) AST also depends on the chemical nature of the phosphorescent compound. Molecules containing carbonyl groups and having nT* lowest triplet states exhibit significantly smaller AST values than aromatic hydrocarbons which have TT* triplet states. In general, AST values for carbonyls are equal to or less than 3000 cm-' (for example, 1530 cm-'
or 41 nm for benzoquinone, 2990 cm-' or 42 nm for formaldehyde), contrasting with large values of AST observed in PAHs. Azaaromatic compounds exhibit AST values that are usually larger (2000 cm-' to 6000 cm-') than those observed with carbonyls. The parameter can therefore be used for differentiating between compounds of various chemical types. The unique aspect of the singlet-triplet selection approach is its ability to obtain in one measurement the phosphorescence spectrum of a specific compound or group of compounds where there is severe overlap because of emission from other components. The depicted signals can be used for qualitative identification as well as for quantitative analysis. T h e preliminary results observed with various P N A compounds are given in Table 11. These data indicate the specificity of the AA values and the applicability of the technique. The preliminary results reported in this study show the methodology is potentially useful for multicomponent phosphorimetric assays. Our discussion reveals the fact that the AST parameter can be a useful distinguishing feature. Interference-free spectra can be obtained from compounds t h a t have the smallest AsT. For compounds that have intermediate values for their AST, the selection of an optimal AX (matching with AsT) should enhance the corresponding synchronous phosphorescence signals, the spectral overlap of other components in the mixture being decreased. Even when the alternate technique of selectively exciting each component in a mixture is possible, several measurements must be made, each using a different excitation wavelength most suitable for one specific component. On the other hand, with the synchronous technique, it is possible to screen out in a single measurement a compound or group of compounds having a given singlet-triplet energy difference, thus reducing the measuring time. By exploiting the singlet-triplet separation differences, this novel approach offers yet another possibility to study selectively specific components in mixtures, which should be useful in those cases where traditional methods using fixed excitation are ineffective.
LITERATURE CITED T. C. O'Haver and W. M. Parker, Anal. Chern., 46, 1886 (1974). G. F. Kirbright and C. D. Delima. Analyst (London), 9 9 , 338 (1974). E. V. Shpolskii, Sov. Phys. Usp., 3 , 372 (1960). G. Mamantov, E. L. Wehry, R. R. Kemmerer, and E. R. Hinton, Anal. Chern., 49, 86 (1977). R. P. Fisher and J. D. Winefordner, Anal. Chern., 44, 948 (1972). J. J. Mousa and J. D. Winefordner, Anal. Chern., 46, 1195 (1974). J. B. F. Lloyd, J . Forensic Sci. SOC.,11, 83 (1971). T. Vo-Dinh, Anal. Chern., 50, 396 (1978). J. Philip and I. Souter, Anal. Chern., 48, 420 (1976). S. L. Wellons, R . A. Paynter, and J. D. Winefordner, Specfrochirn. Acta, Pan' A , 3 0 , 2133 (1974). T. Vo-Dinh, E. Lue Yen, and J. D. Winefordner. Anal Chern.. 4 8 , 1186 (1976) - -, T. Vc+Dinh, E. Lue Yen, and J. D. Winefordner, Talanta, 2 4 , 146 (1977). T. Vo-Dinh, G. Walden, and J. D. Winefordner, Anal. Chern.. 4 9 , 1126 (1977). T. Vo-Dinh and J. D. Winefordner, Appl. Spectrosc. Rev.. 13 (2), 261 (1977). A. P. Swain, J. E. Cooper, and R. L. Stedrnan, Cancer Res., 2 9 , 579 (1969). C. E. Searle, Ed., "Chemical Carcinogens", ACS Monogr., 1 7 3 , Library of Congress, Washington, D.C., 1976, p 253. Coal Technology Program-Annual Interim Report for fiscal year ending June 30, 1976, Oak Ridge National Laboratory, 1976, p 96. T. Vo-Dinh and J. R. Hooyman, to be published. J. B. Birks, "Photophysics of Aromatic Molecules", J. Wiley & Sons, New York, 1970. S.P. McGlyn, T. Azurni, and M. Kimoshita. "The Triplet State", Prentice Hall, New York. 1970. \
RECEIVED for review May 3, 1978. Accepted September 11, 1978. Research sponsored by the Division of Biomedical and Environmental Research, U.S. Department of Energy under contract W-7405-eng-26 with the Union Carbide Corp.
