(11) Paul C . Dryden, Ph.D. Thesis, University of Illinois, Urbana, Ill., 1975. (12) E. D. Cehelnik and K . D. Mieienz, Appl. Opt., 15, 2259 (1976). (13) Melton F. Bryant, PhD. Thesis, University of Illinois, Urbana, Iii., 1977.
RECEIVED for review April 4, 1977. Accepted June 29, 1977.
One of the authors (J.A.P.) expresses his appreciation for a Uniroyal Fellowship for part Of this and the authors are grateful for partial support of the work by the NIH under Grant H E W PHS GM 21984, and by the N S F under Grant N S F M P S 74-12248.
Prediction of Peak Wavelengths and Intensities in Synchronously Excited Fluorescence Emission Spectra J. B. F. Lloyd" and I. W. Evett Home Office Forensic Science Laboratory, Priory House, Gooch Street North, Birmingham 85 6 0 0 ,
Peak wavelengths and Intensity maxima of synchronously excited fluorescence emission spectra of 33 compounds are computed for various excitation intervals (OX) on the assumption that the spectra can be represented as the products of single Gaussians, in reciprocal wavelengths, parameterized from the experlmental excltation and emission spectra. Over the range of has,,, (273-594 nm) and of D h (1.5-330 nm) covered, the mean dlfference between calculated and observed values of is 1.95 nm; the maximum dlfference Is 14 nm. The corresponding values for the intensities are 0.063 and 0.348 relative to maxlmum intensities of 1.0. An approximation from the product Gaussian gives: = 2 hoeXhoe,,,(hoex hoem OX) where Xoex and hoe,,, are the peak wavelengths of the experlmental excitation and emission spectra. From this relationship, Xos,emvalues may be calculated to an accuracy negligibly less than that of the full equation over the range of a compound's peak half-width.
+
-
Since the introduction of the technique ( I ) , synchronously excited fluorescence emission spectra have enabled traces of fluorescent materials to be characterized for forensic purposes in a wide variety of cases ( Z ) , and have also found application in the field of oil pollution ( 3 , 4 ) . Despite the claim that the factors influencing the technique have been elucidated (31, t o date the characteristics of the spectra have been considered only in qualitative terms ( 5 ) . We wish to show that the characteristics of the spectra can be derived quantitatively from the characteristics of the corresponding fixed excitation/emission spectra. EXPERIMENTAL The fluorescence spectra, which are linear in wavelength, were recorded with a Perkin-Elmer MPF-4 spectrofluorimeter operated in the "true" excitation mode, whereby the spectra are automatically compensated for the spectral distribution curve of the source photon output and of the excitation optics. The spectral bandpass was set at 3 nm, except for the polynuclear hydrocarbons for which a setting of 0.5 nm was used. All of the organic compounds (Table I) were at least reagent grade in quality. The samples of them used, except riboflavin, contained less than 1YOof extraneous fluorescence at the relevant peak excitation and emission wavelengths so far as could be detected by TLC (silica gel with appropriate solvent mixtures chosen from n-hexane, ethyl acetate, acetic acid, and water) or by HPLC (19-cm column of ODs-Partisil5 in aqueous methanol). The sample of riboflavin was purified by HPLC. Cerous sulfate 1710
ANALYTICAL CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
U.K.
was of analytical grade, with which its fluorescence excitation and emission spectra were in agreement. Solutions were prepared in methanol and/or water, neither of which contained fluorescent impurities detectable at the maximum sensitivities used. All the solutions were diluted to give absorbance values not greater than 0.01 over the pertinent wavelength range, except for benzo[alpyrene, where the value was 0.06 because of the high detection limits imposed by the narrow bandpass used. The solutions were not de-aerated. Computations were made on the Honeywell time-sharing network. THEORY For the present, we restrict discussion t o single-peaked excitation and emission spectra. T h e signal response (R,J observed a t a given wavelength (Aex) in a fluorescence excitation spectrum, relative t o the response (Rhoex)a t t h e of maximum excitation efficiency, is repwavelength (A),' resented as a Gaussian in reciprocal wavelength (6). (This point is discussed subsequently). Hence:
where ueXis the standard deviation of the Gaussian, in units of reciprocal wavelength. The emission spectrum is similarly represented, with all terms carrying subscript em. At a given point (As,em) in a synchronously excited emission spectrum, the observed fluorescence signal ( R A J will be a product of the relative response a t ,A, in the fluorescence emission spectrum and the relative response (Le., relative excitation efficiency) a t A,, in the excitation spectrum: -1
'lR'em
'''ern
Under usual conditions Rp,, = Rxo,,, because each is the same detector response to the same fluorescence excited a t Aoex and Therefore Rx,,,, varies between 0 and 1 monitored a t.,,A' relative t o Rho,,. If the synchronous excitation interval, separating excitation and emission wavelengths by a constant amount in a given spectrum, is represented by DX,substitution of Equation 1 and the analogous equation for the emission spectrum into Equation 2 gives: Rhs,em = e x P C - [ ( X s , e m - '
- [(A,,,,
- hoe,-'
-'I
- D h ) - ' - A 0ex
I
i20emzI
/20ex2)
(3)
where ,A, has been substituted for ,A, t o indicate a synchronously excited emission is now concerned.
Table I. Experimental Fluorescence Parameters of Various Compounds Parameters, nma Compound
''em
''ex
a 'em
A 'ex
Acridine oraiige 49 47 521 489 Anthranilic acid 390 319.5 58 2 51 403.6 363.6 13 3 5 k n z o [ a Ipyrene 363 Benzo[f]quino66 4 47 427 lineb Cerous nitrateC 53 5 353 25 2 29 2,6-Di-tert-butyl6 303 33 27 27 8 4-methyl phenol 1,3-Dihydroxy7 371 24 235 47 naphthalene 61 3,3'-Dimethoxy376 50 300 8 benzidine Diphenylamine 355 51 35 284 9 Diphenyl ether 10 29 2 270 27 27 11 Eosin 34 29 541 5 24 L-Ephedrine 25 8 27 20 12 281 Erythrosin B 13 549 35 30 530 (Na salt) 4-Ethyl-7-di14 54 450 374 56 methylaminocoumarin 15 Fluorescein (Na 34 31 517 496 salt) 16 3-Hydroxy-25 09 352 98 48 naphthoic acid (A1 complex) 4,4'-Isopropylidene- 302 17 27 8 30 25 diphenol 1-Naphthold 18 294.5 62 52 356 2-Naphthold 41 7 347 60 45 19 1-Naphthylamine 423 319.5 72 55 20 61 1-Naphthylamine404 329 52 21 4-sulfonic acidd Perylene 22 438.8 435.5 12 9 p-Phenylenedi43 23 39 4 309 53 amine 31 2-Phenylphenol 44 24 332 28 8 Quinine sulfateb 446 50 25 351 79 Rhodamine B 26 568 544 36 35 31 Ribof lavine 27 521 26 8 74 36 Salicylamide 28 428 304 72 29 298 31 Salicylic acidd 400 60 30 334 44 Sulfanilamide 26 2 28 31 41 Sulfanilic acid 332 253 30 364 56 73 1-(p-Sulfonamido- 438 32 phenylF3-Cpchlorop heny1)A *-pyrazoline 33 Thioxanthen-953 429 379 42 one In a Meanings of the symbols are given in the text. acidic (0.01 M H,SO,) MeOH. In water. In alkaline (0.01 M NaOH) MeOH. e In aqueous MeOH. All other solutions are in MeOH. 1
X inrnl
Fluorescence spectra of diphenylamine in methanol. (A) Excitation monitored at 355 nm; (B) emission excited at 284 nm; (C) synchronously excited emission with intervals of 30, 70, and 100 nm in order of the peak maxima. Full lines are the experimental spectra. Crosses and filled circles indicate points on the respective Gaussian and Lorentzian profiles parameterized, in reciprocal wavelength units, from the experimental spectra. Open circles are points on the product Gaussian (Equation 3) Figure 1.
T h e required standard deviations are obtained from the of the exciexperimental peak half-widths (AXexand AX), tation and emission spectra as follows. At the half peak height it may be derived directly from Equation 1 that:
which yields a positive root for
uex(and
oeX= { [ 1 + ( A X e x / h o e x ) 2 ]
similarly for
of
gem)
- 11 x
[ A h e x ( 2In 2 ) 1 ' 2 -' ]
(5)
Hence, uexand gemare obtained when the appropriate experimental values from the excitation and emission spectra are inserted into the equation. An approximation for the relationship of to X'ext XOem and DX can be derived from Equation 3. Without loss of generality, we make the transformation: =
(1+ l3-'
,A,"
where B is a "dummy" variable introduced to facilitate the subsequent manipulation. Substitution of Equation 6 into Equation 3, and solving for dRx,,,, / d B = 0 yields:
BO
oex
-2
2-
'em
'em
'Oem
2
[Xoem - D X ( 1 + B o ) ] ' +
1 + B" [ioem - (1+ B")
1
-
E]
(7)
=
where Bo is the value of B corresponding to the wavelength of maximum intensity in a synchronously excited emission spectrum. Generally DX is small with respect to Xoem, and Bo lies between *0.1. We therefore neglect terms in BoDX. If (Xoem - DX)/Xoem= y , rearrangement of Equation 7 with substitution of Bo as defined in Equation 6 yields:
where is the wavelength of maximum intensity in the synchronous spectrum. Typically, y lies between 0.9 and 1.0, and when emission and excitation spectra represent transitions between the same electronic states, crex = uem, as their usual mirror symmetry relationship requires. Hence, the term in y and u may be approximated to 2. This substitution, and rearrangement, gives: Xos,em =
2PemA",, (A",,
+ Pex - m)-'
RESULTS AND DISCUSSION The fluorescence parameters of the compounds used to test the above relationships are given in Table I. All of the compounds examined in the present connection are included in the Table, except for those found to be either too photolabile or too weakly fluorescent to enable accurate measurements to be made. We have compared t h e experimental excitation and emission spectra of ten of the compounds with Gaussian profiles (Equation 1) parameterized with the experimental values of Xoex, ioem, uexand gemtaken from Table I. (U-values are calculated from AX -values by means of Equation 5 ) . A typical comparison, in the case of diphenylamine, is illustrated a t A and B of Figure 1, where the crosses indicate points calculated with Equation 1. In this, and in the other cases ANALYTICAL CHEMISTRY, VOL. 49,NO. 12, OCTOBER 1977
1711
examined, there are no marked consistent differences between t h e Gaussian and experimental spectra except in the short-wave regions of the excitation spectra, where the experimental response generally exceeds the Gaussian. This is expected, because whereas an emission spectrum generally corresponds to transitions only from the first excited state, the short-wave side of an excitation spectrum may contain contributions from transitions to higher states. Experimental spectra are subject to variations imposed by the characteristics of the spectrofluorimeter used; for instance, by variation in the spectral intensity distribution over the bandpass used, and by variation with wavelength of the source output, of detector sensitivity, and of the transmittances of beam splitters and monochromators. The major variation likely to be imposed on the spectra will be derived from the characteristics of the excitation source. All of the data used in this work are taken from spectra that were automatically corrected for this effect, and for the others to which excitation spectra are subject, by our spectrophotofluorimeter. The emission spectra are uncorrected, but over the wavelength range of each fluorescence emission we find that the instrument's spectral sensitivity distribution does not vary to a n extent sufficient to affect significantly measured Xoemvalues. Some distortion of emission band shapes might occur, which could affect o,,-values. However, provided that the Gaussian band-shape is retained, the effect affects the synchronously excited emission spectra also, and is therefore self-canceling so far as comparisons between the calculated and the experimental spectra are concerned. We have examined the possibility that the spectra should be represented by a Lorentzian distribution; i.e. that:
in the case of the excitation spectrum, where hex is the peak half width of the spectrum presented in units linear in reciprocal wavelength. T h e calculated Lorentzian intensities (given Xo- and Am-values from the experimental spectra) for the excitation and emission spectra of diphenylamine are included in Figure 1 (filled circles). All of the ten compounds examined gave similar results: relative to the Gaussian the slightly "waisted" nature of the Lorentzians and their extensive tailing resulted in depreciated agreement with the experimental spectra. Neither is it apparent that the spectra might be usefully represented by a sum or product of the two distributions, e.g., the Voigt function (7, 8), either in terms of mathematical convenience or of improvement to the fit provided by the generally satisfactory performance of the Gaussian. Although in nine out of ten of the examples examined, the experimental curve in the long-wave region of the emission lay between the Gaussian and the Lorentzian, as in Figure 1, in the long-wave region of the excitation, the Gaussian intensities were always closer to the experimental, and were invariably exceeded by the Lorentzian. Again, Figure 1 is typical. Evidently, a combination of the two functions could only improve agreement in one part of a spectrum a t t h e expense of worse agreement in another. Although the processes involved in broadening excitation and emission bands may individually give rise to Lorentzian distributions (7, 8), the combination of many such distributions, arising from the many vibrational and rotational sub-features present in the spectra of complex and variously solvated molecules, will result in Gaussian distributions (7), in agreement with our result. For each compound in Table I, the values of h',,,, maximizing R , at various values of Dh were calculated by means of Equation 3 in conjunction with a n iterative computer program for location of the peak maxima written by J. M. 1712
ANALYTICAL CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
Dubery. The experimental data used for the calculations are listed in Table I. Equation 5 was used to calculate values of u,, and uemfrom Ah,, and AXem. These results, for calculated synchronously excited spectra, are compared with data taken from experimentally plotted spectra in 'Table 11. The experimental results for each compound are from a synchronously excited emission attributable to the observable range of a single excitation and a single emission peak within the limits that outlying regions, corresponding to less than 3 70 of the maximum fluorescence intensity, are excluded. Where several excitation peaks were available, the longest wavelength peak was normally used, although in some cases, e.g., riboflavin, other peaks have been used either to extend the excitation interval over which comparisons could be made or to increase the intensity of the spectra. Because the distribution of experimental from calculated results appears to be non-Gaussian, the variation between the two sets is presented, in Table 11, in terms of mean and maximum deviation. Some of the calculated results for the synchronously excited emission spectra of diphenylamine are plotted, as open circles, on the experimental curve in Figure 1 (C). All of the maximum deviations of Xns,em and of the corresponding values of RLrm(i.e. RXoap,)recorded in Table I1 occur towards the extremities of the spectra, an expected consequence of the use of single Gaussians. In some cases, new peaks emerge a t the extremities as indicated in the Xns.em column of the Table; or the extremities of the experimental spectra may be significantly intensified as in the illustrated (Figure 1)case of diphenylamine a t an excitation interval of 100 nm. The particularly large variation in RxoB,,,in the case of benzo[a]pyrene (compound 3) occurs a t a point (404 nm) where considerable intensity must be contributed by a n excitation (at 346 nm) overlapping the relatively broad one a t 363.6 nm used in the calculation. Perylene (compound 2 2 ) is a similar case. Overall, however, and particularly in view of the wide range of excitation intervals (1.5-330 nm) and emissions (273-594 nm) covered, the mean difference between calculated and experimental wavelengths of 1.95 nm, and between calculated and experimental intensities of 0.063, taken over 353 spectra, supports the view that the assumptions made are not without justification and are a satisfactory basis on which the characteristics of synchronously excited emission spectra may be predicted. The calculations require access to a computer. Although a useful rule-of-thumb follows from the experimental observation of a seemingly roughly linear dependence of on DX when small segments of the spectral ranges are considered-an increase in DX by 10 nm generally increases by about half that amount-lines of attempted linear regression do not pass through the point Xos,em = Anem when DX = Anem - ioex, as they should. More satisfactory answers are provided by the derived hyperbolic, Equation 9. We have tested the validity of the assumptions made by writing the equation in the form:
which is obtained by rearrangement and making the substitution C = (2 XoeX)-', and examining the rank correlation of C (calculated individually from the experimental data for each of the 33 compounds under synchronous excitation conditions at all the values of DX on which Table 11 is based) on the actual values of The Spearman correlation coefficient (9) obtained, 0.847, is very highly significant. A linear regression analysis of C on Xn,L1 yields C = 0.496 XoeL1 + 0.0517.10-3, in agreement with the value required by Equation 11, i.e., C = 0.5 A comparison between the performance of the product Gaussian (Equation 3) and the hyperbolic (Equation 9) in
Table 11. Comparison of Experimental Data from Synchronously Excited Fluorescence Spectra with Data Calculated from the Product Gaussian Equation 3 Experimental synchronous excitation data useda Compoundb 1
2 3 4 5 6 7 8 9 10 11
12 13 14 15 16
17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Overall:
---
Deviation of calculated from experimental resultsf hos,em?nm
-
Range of '"s,em nm 509- 550 362-442 403.2-404.1 398-464 323-370 294-338 344-419 356-422 330-405 287-301 537-581 27 3-294 541-566 419-490 509-560 451-590 295-327 341-400 377-461 383-486 377-453 437.5-446.5 368-428 315-369 392-490 558-581 491-594 386-499 366-466 321-374 317-370 401-467 409-467
Range of D h , nm
No of Dh'sC
Mean deviation
Max deviation
Mean deviation
Max deviation
10-70 10-140 34-50 25-120 60-120 7-70 100-190 20-150 20-1 30 10-40 10-60 10-45 5-45 20-140 6-70 80-250 10-60 40-120 10-130 40-180 20-150 1.5- 20 35-135 10-90 20-150 5-50 210-330 40-200 50-180 50-120 50-130 10-1 20 15-110
7 13 14
2.4 1.4 0.41 3.9
10
1.8
9 15 14 12 5 6
1.9 0.8 2.9 1.9
7.1d 3.8 0.88 5.8 2.6 3.5 1.5 8.4 4.1 2.2 12.'id 2.4
0.039 0.060 0.146 0.087 0.054 0.065 0.055 0.138 0.029 0.032 0.049 0.103 0.018 0.067 0.061 0.024 0.033 0.055 0.04 5 0.072 0.086
0.122 0.167 0.324 0.221 0.129 0.144 0.152 0.221 0.064 0.073 0.141 0.238 0.026 0.127
273-594
1.5-330
8
8
6 13 9 10 11
9 12 15 14 9 10
9 14 7 12 17 14 8 8 12 13 353
1.1
2.8 1.2 0.5 2.3 1.9 1.3 0.8
1.1
1.2 0.93 1.3 1.3
7.7 12.8d 3.4 1.4 3.4 8.5 7.9 5.4 3.4 1.8 3.5 10.I d 1.5 3.7 14.0d 2.7 2.0 1.8 4.2 3.0
1.95
14.0
1.8 2.1
3.3 3.0 1.56 0.93 2.1 2.6 0.8 1.8
4.3 1.3
0.160
0.081
0.101 0.082 0.087 0.119 0.132 0.167 0.348 0.087 0.195 0.103 0.046 0.221 0.076 0.162 0.081 0.193 0.133 0.165
0.0634
0.348
0.117
0.047 0.077 0.038 0.021 0.094 0.020 0.054 0.047 0.100
0.054
Additional to the data in Table I. Identities are given in Table I. Excluding the value Dh = haem - hoex. Subsidiary peaks present at the extremities of D h . e Rhose varies from 0 t o 1. f The calculated results are from Equations 3 and 5 parameterized with the experimental dati%om the excitation and emission spectra listed in Table L The experimental results are measured directly from the synchronously excited emission spectra. a
terms of the range of experimental values for each compound that can be matched to within 5.4 nm by the results calculated from these equations for various values of Dh is is shown in Table 111. (As previously mentioned, ,A,' computed from the Gaussian by finding the value of A,, which maximizes Rks,em). In both cases, the overall range is of the order of a peak half-width of the fluorescence emission spectra, of which the values (AXem) are given in Table I, i.e., over most of the intensity range that is likely to be useful in analytical work. T h e only major disagreement with this generalization occurs with 3,3'-dimethoxybenzidine (compound 8 ) , which is undoubtedly due to the approximation made in deriving Equation 9-that u,, and uem do not significantly differ from one another. When the actual values of 0.000285 and 0.000150 nm-' respectively (calculated with Equation 5 ) are used in Equation 8, with the assumption that y approximates to unity, the defined spectral range becomes 32 nm, i.e., only 4 nm less than the result from the product Gaussian. The only other example, among those in Table 111, of values of uexand uem widely differing from each other, is benzo[alpyrene (compound 3) where the values are 0.0000417 and 0.0000130 nm-l. For this compound, the limitations imposed by other transitions on the spectral range that can be examined do not permit realistic comparisons t o be made between the calculated data, and their agreement with exper-
iment, a t values of DX extended to the point where large deviations in might occur. It was shown that much improved agreement with experiment did occur, however, when u-values were used in conjunction with Equation 8 (with y = 1) to calculate T h e mean deviation from experimental values became 0.3 nm, in contrast to the value of 2.6 nm shown in the Table. Indeed, the agreement a t every one of the eight excitation intervals examined was superior to that obtained from the product Gaussian. For the remaining 31 compounds, the assumption of equal u's in the deviation of Equation 9 seems to be experimentally justified. The mean value of uex/uemo f t h e compounds is 1.10. The standard deviation of the ratio is 0.179. In general, unless there is an obviously exceptional difference between excitation and emission peak half-widths, e.g., corresponding to uex/uem > 1.5, there seems little to be gained from the use of Equation 8 in preference to Equation 9. An important attribute of the synchronously excited emission spectra is their experimentally observed, much reduced peak half-widths (represented as relative t o their originating excitation and emission spectra. In the present series of compounds (Table I), the overall mean values of AX,,e,, AX, and AX, were found to be 28.4, 38.3, and 49.9 nm respectively, under the condition that DX = ,,A' - Xoex. Above and below this value of D h , Ah,,,, is observed to increase and decrease respectively. We have not derived a ANALYTICAL CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
1713
Table 111. Comparison of Results Calculated from the Product Gaussian and Hyperbolic Expressions with Experimental Results Gaussian (Eq. 3 ) Hyperbolic (Eq. 9 ) Spectral Mean devia. Suectral Mean deviarange ( n m ) tion within range (nm) tion within Com- f o r 5 . 4 nm range (nm) for 5.4 nm range (nm) pound accuracya (No. in set) accuracya (No. in set) 1 1.6 ( 6 ) 32b 1.6 ( 6 ) 32 1.4 (13) 2.3 (11) 2 80b 71 0.9b 0.9b 2.6 ( 8 ) 3 0.4 (8) 2.4 (11) 4 52 29 2.3 ( 7 ) 5 1.8 (10) 47b 3.0 (10) 47b 44b 6 34 1.9 ( 9 ) 2.5 ( 8 ) 75b 0.7 ( 1 5 ) 7 7 5b 0.8 ( 1 5 ) 36 2.0 ( 3 ) 8 6 2.2 ( 1 0 ) 2.1 (10) 1.9 ( 1 2 ) 7 5b 9 52 14b 14b 1.1 ( 5 ) 10 1.2 (5) 11 19 19 1.2 ( 4 ) 0.4 ( 4 ) 1.3 (8) 21b 12 21; 1 . 2 (8) 13 25 0.5 ( 6 ) 25’ 0.9 ( 6 ) 14 1.1( 9 ) 41 1.6 ( 1 0 ) 47 15 29 1.4 ( 7 ) 1.0 ( 7 ) 29 1 . 3 (10) 16 1.5 (8) 13gb 108 17 32b 3 2b 0.8 (11) 1.8 (11) 18 596 1.8 (9) 59b 1.9 ( 9 ) 2.0 ( 1 0 ) 19 69 1.0 ( 1 0 ) 69 2.1 (15) 2.5 ( 1 2 ) 20 70 91 21 76b 3.0 (14) 76b 2.8 ( 1 4 ) 22 1.4 ( 9 ) 9b 1.6 ( 9 ) 9b 23 0.9 ( 1 0 ) 60b 1.9 ( 1 0 ) 60b 24 2.1 ( 9 ) 29 54b 1.2 ( 6 ) 25 2.0 ( 1 3 ) 62 1.6 ( 1 0 ) 88 26 0.8 ( 7 ) 23b 0.4 ( 7 ) 23 27 1.8 ( 1 2 ) 53 2.2 ( 6 ) 103b 28 2.3 ( 1 3 ) 56 100 1.7 ( 9 ) 29 66 2.3 (11) 100; 1.3 ( 1 4 ) 30 44 53 2.0 ( 7 ) 1.2 ( 8 ) 31 53b 0.9 ( 8 ) 53b 1.4 (8) 66b 1.3 (12) 66b 1.1( 1 2 ) 32 33 1.9 ( 1 3 ) 1.3 ( 1 3 ) 58 b 58b Means:
53.9
1.51 (323)
46.6
Table IV. Comparison between Calculated ( Hy?erbolic b u a t i o n 9 ) and ExDerimental Waveleneths of Sy‘nchronously Excited Peaks of Soiiie Anthracene ‘La Transitions Peaka
Dh,
nm
Experimental, nm
Calculated, nm 376.7 37 8.1 380.7 370.9 376.1 395.3 372.1 381.4 401.4 377.6 396.5 417.2 374.6 383.3 392.3 402.2 422.8 380.5 398.1 417.6 378.8 394.9 404.1 423.5 445.2 401.1 419.2 451.2 415.8 425.4 445.8 422.2 441 .O 452.1
la la la 2a
2 5 10
376.7 378.5 382.4
10
2a lb 3a 2a lb 3a 2b
20 20 30 30 30 40 40 40 50 50 50 50 50 60 60 60 70 70 70 70 70
lb
IC
4a 3a
3b 2b I C
4a
3b 2c 5a 4b
3b 2c Id 4b 3c Id 4c 3c 2d 4c 3d 2d
80
80 80 90 90 90 100 100
100
376.1 395.3 shC
380.6 403.1 377.4 396.3 414.8 376.7 382.5
lb
400.9 424.1 379.5 398.2 416.3 377.5
Ib
402.9 424.1 444.5 400.1 419.1 453.1 425.3 445.8 422.1 439.0 452.3
Ah,,,=.,
=
0.364 (Ah,,
+ Ah,,
) - 3.309
when the above condition applies. The corresponding correlation coefficient is 0.970. Qualitatively, the dependence of on DX can be seen (e.g., with reference to Figure 1) to arise because at low values of DX, the synchronously excited emission intensity derives from adjacent regions of the excitation and emission spectra where the intensity varies with wavelength very much more rapidly than in the regions “seen” by each spectrum beyond the maximum of the other when DX > Xoem - hoex. We now consider the multi-peaked spectra produced on occasions by single compounds. The hyperbolic approximation provides a ready means of analyzing the sometimes complex spectra that the technique can produce under some conditions. Anthracene is taken as an example, and we refer to the four most intense vibronic components of the excitation and emission spectra derived from the ‘La state. (The weak fine structure associated with these transitions affects the spectrum 1714
ANALYTICAL CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
+1.7 I 0.0 0.0
I 0.8 + 1.7 -0.2 -0.2 - 2.4 + 2.1 - 0.8 I - 1.3 1-1.3 -1.0 + 0.1 - 1.3 -1.3 I --1.2 + 0.6
-
- 0.7
-1.0 -0.1
+ 1.9 I
- 0.1 0.0 -0.1
2.0 +0.2
-
0.84
(of modulus)
Max. difference
a
satisfactory approximation from the product Gaussian (Equation 3) for these quantities (they could, of course, be derived by iterative computation of the pairs of X,,,,-values corresponding to Rx,:e, = 0.5 in Equation 3), but by linear regression our experimer.ta1 results yield the empirical relationship:
0.0
+ 0.4
Mean
1.80 (297)
The range is the experimental one over which the calculated peak wavelengths were in agreement with experiment to the indicated level of accuracy. In these cases none of the data available exceeded 5.4 nm.
lb
Difference, nm
2.4
No. 1-4 represent excitations at 374.1, 355.2, 338.0, and 322.8 nm; letters a-d are emissions at 377.1, 398.2,
421.1, and 446.7 nm.
Not detected.
Shoulder.
only slightly, and is not further considered.) Each emission is subject to four excitations, therefore, at a given value of DX, 16 transitions might be observed. Most of these will be extremely weak, and we restrict discussion to the transitions expected to arise from combinations of excitation and emission bands lying within the interval of DX f 14 nm from one another (the greatest AX-value of the excitation and emission bands is 14 nm). It follows from the Gaussian equations that this restriction excludes from consideration the synchronously excited emissions less intense than about 7.5% of the maximum possible intensity. A comparison between the wavelengths of the observed peaks and the wavelengths calculated by means of Equation 9, taken over 1 2 values of DX, is shown in Table IV. Of the 34 peaks predicted to be present, 29 are found. The outstanding five peaks remain buried in accompanying peaks derived from the more intense excitation and emission components. Because of the accuracy with which the peak positions are predicted, as the Table shows, all the resolved peaks can be unambiguously assigned. The significance of the above considerations to the choice of conditions for “fingerprinting” samples of fluorescent mixtures is illustrated with reference to Figure 2, which shows spectra obtained from a cyclohexane extract of some fragments of soot. Spectrum A was synchronously excited at an interval of 19 nm, to obtain the maximum amount of spectral detail.
Table V . Fluorescence Characteristics, in Cyclohexane, and Predicted Values of
h s.em,
of Some Polynuclear Hydrocarbons
Predict e d
hos.em a
DA
Compound
nm
Benzo[k]fluoranthene Benzo [ a Ipyrene Perylene Anthanthrene
362 ( - ) , b 3 8 2 (lo), 401 ( 1 4 ) . 346 (-), 364 ( 1 5 ) , 384 (13). 386 ( - j, 407 (19 ), 429 ( 1 2 ) , 4 3 5 ( 1 2 ) . 403 (14),c 421 ( 5 ) , 429 ( 4 ) .
1 9 nm
403 ( 5 ) , 427 ( B ) , 453 ( 8 ) . 403 ( 5 ) , 427 ( B ) , 454 ( 8 ) . 4 3 8 (12); 4 6 5 ( 1 4 ) , 497 (-). 4 3 0 ( 4 ) , 435 (-), 457 (10).
403, 425. 403. 433, 4 4 1. 429, 4:37, 449.
1.5 nm 403.
none 438. 430.
a Calculated b y means of Equations 5 and 8 from the ho and hoe data, under the restrictions given in the text. Parentheses enclose values of Ah,, or Ah,,; in the instances engyosing a &sh, the peaks concerned were extensively overlapped by adjacent peaks. The two, almost entirely overlapping, peaks centered at this point were taken together.
I
103
122
L30
A
B
1
3 65
500 h inrl
Figure 2. Synchronously excited fluorescence emission spectra o f a cyclohexane extract of some soot particles. The excitation intervals are (A) 19 nm and (B) 1.5 nm. The numbers on the spectra are the wavelengths (nm) of the proximate p e a k s
Fewer peaks were present, or less well resolved, a t other excitation intervals. Although spectrum A might consequently be considered to be the most valuable fingerprint that could be obtained, it is in fact a misleading representation of the composition of the mixture, as more detailed consideration shows. Most of the fluorescence of this type of extract is due to the compounds benzo[k]fluoranthene, benzo[a]pyrene, perylene, and anthanthrene, although many other less prominently fluorescent components are also present (10). Our data for the excitation and emission bands of the four named compounds, in the region above 350 nm are listed in Table V. Only the prominent bands, in excess of 15% of the most intense in each case, are listed. Hence, from Equation 2, synchronously excited emissions of the individual compounds of a relative intensity less than (15/100)2, i.e. 0.0225, under optimum excitation conditions, are excluded from consideration. As U-values vary widely in these particular compounds, the individually measured value is used for each excitation and emission peak, and the predicted values of Xos,em are calculated with Equation 8. As before, only synchronously excited emissions expected from pairs of excitation and emission peaks lying within DX k AXemor DX f Ah,,, whichever is the greater, are considered. These predicted values are collected in Table V. Comparison between them and the observed values indicated alongside the relevant peaks in Figure 2A immediately reveals that most of the fluorescence in this spectrum is derived from a single compound, Le., anthanthrene. If any perylene is present it is not detected (in this case, the separations between the pairs of peaks considered in the calculations of are near the extremities of DX f AX,,, therefore detection limits are high); and a t 403 nm, benzo[a]pyrene and benzo[k]fluoranthene entirely
overlap. Only the latter compound is separated from the group, a t 422 nm, although the difference between this and the predicted value (425 nm) suggests that interfering compounds are present. From Table V, it is apparent that with this particular set of compounds, the most satisfactory “fingerprint”, containing distinguishable contributions from as many anticipated compounds as possible, would be obtained a t excitation intervals corresponding to the compounds’ rather small Stokes shifts, i.e., 1-3 nm. Except for benzo[a]pyrene, each exhibits a strong 0,O transition in both excitation and emission. Under such conditions, the term Anem - Xoex - DX in Equation 8 becomes negligible and Xos,em XO,,,,. The actual spectrum of the solution excited a t an interval of 1.5 nm is shown a t Figure 2B, where the three indicated emission wavelengths correlate identically with those in Table V. Perylene is now clearly seen, although at a relatively low level, at 438 nm, and likewise benzo[k]fluoranthene, at 403 nm. Benzo[a]pyrene does not significantly contribute to the spectrum; its presence follows from the difference between the 403 and 422 nm emissions in spectrum A. If only benzo[k]fluoranthene were present, these would be approximately equal in intensity ( I ) . In only the latter respect is any useful information present in spectrum A. Finally, this example of the application of the derived equations is pertinent to the characterization of fluorescent mixtures generally, given that adequate qualitative information on their possible components is available. The choice of experimental “fingerprinting” conditions on, alternatively, a “hit-or-miss” basis, without reference to the characteristics of the compounds concerned, is unlikely to exploit adequately the considerable potentialities of the technique, and may well produce ambiguous results.
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ACKNOWLEDGMENT We are very much indebted to J. M. Dubery for his help with the computation. LITERATURE C I T E D (1) (2) (3) (4)
J. B. F. Lloyd, Nature (London), Phys. Sci., 231 64 (1971). J. B. F. Lloyd, Chem. B r . , 11, 442 (1975). P. John and I. Soutar, Anal. Chem.. 48, 520 (1976). D. C. Gordon, Jr., P. D. Keizer, W. R. Hardstaff, and D. G. Aidous. Environ. Sci. Techno/., 10, 580 (1976).
(5) J. E. F. Lloyd, Analyst, (London),100, 82 (1975): and references therein. (6) C. Sandorfy, “Electronic Spectra and Quantum Chemistry”, PrenticaHall, \‘nc., Englewood Cliffs, N.J.. 1964. Chap. 5. pp 104-105. (7) J. I. Steinfeld. “Molecules and Radiation”, Harper and Row, Inc., New York, N.Y., 1974, Chap. 1, pp 22-24, and references therein. (8) J. D. Winefordner, S. G. Schulman, and T. C. O’Haver, “Luminescence Soectrometrv in Analytical Chemistry”, Wilev-Interscience. Inc.. New York. N.Y., i972, Chap. 2, pp 25-30. (9) M. G. Kendall, “Rank Correlation Methods”. 2nd ed.. Charles Griffin and Go. Ltd.. London. 1955. Chao. 1. (10) J. B. F. Lloyd, J . Forensic Ski. Soc.,11, 235 (1971)
RECEIVED for review March 22 1977. .Accepted July 13, 1977. ANALYTICAL CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
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