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Anal. Chem. 1981, 53, 1244-1249
(12) Lindhard, J.; Scharff, M.; Schiott, H. E. Mat.-Fys. Medd.-K. Dan. Vidensk. sekk. 1963, 33 no. 14; Chem. Ab&. 1964, 60, 13898e.
RECEIVED for review March 2,1981. Accepted April 22,1981. The Rockefeller portion of this work was supported in part
by a grant from the Division of Research Resources, National Institutes of Health, The Manitoba portion was supported by a grant from the Natural Sciences and Engineering Research Council Canada, which also provided a postgraduate scholarship for W.E.
Fluorescence Photoselection of Matrix Isolated Polycyclic Aromatic Hydrocarbons Jon R. Maple“ Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131
E. L. Wehry Department of Chemistry, Universm of Tennessee, Knoxville, Tennessee 3 79 16
The analytlcal utility of fluorescence photoselection for dlstlngulshlng overlapping spectral bands In mixtures of matrix Isolated polycyclic aromatlc hydrocarbons Is explored. The use of a polarizer to selectively pass the fluorescence of one component from a mixture of fluorophores afler excltatlon by a polarlzed (laser) source results In slgnlficantly Improved spectral dlscrlmlnatlon. Potentlal applications, as well as the limits of appllcablllty, are discussed.
The detection of individual polycyclic aromatic hydrocarbons (PAHs)in complex samples, such as “synthetic fuels”, represents a challenging problem to the analytical chemist. Recently, highly sensitive low-temperature fluorometric procedures (1-B), coupled with laser excitation (2-B), have been successfully exploited for the analysis of complex mixtures of trace amounts of PAHs and derivatives. Selective excitation (2-41, site selection (5-7), and time resolution (7, 8) techniques have proven especially effective as analytical procedures, providing a high degree of selectivity. However, because there are numerous situations in which these methodologies cannot be applied or are of limited usefulness, we have explored the use of photoselection techniques for improvement of selectivity in the low-temperature fluorometric characterization of multicomponent samples. Although it is well-known that the fluorescence from PAHs in rigid media is partially polarized, especially when a polarized excitation source is used (e.g., a laser), the analytical advantages that can be extracted from this phenomenon have not been delineated. In a mixture in which the fluorescence from each compound is preferentially polarized in different directions, enhanced spectral selectivity for a particular compound intuitively may be expected if a polarizer is oriented to selectively pass the emission from the component of interest. However, in order to intelligently interpret or utilize the phenomenon of polarized emission and absorption, the results of the theory which is used to interpret experimental measurements of the polarization of electronic transitions must be understood. In general, the theories assume that the sample molecules either are preferentially oriented in one direction or are oriented randomly (9). Samples which are prepared by matrix isolation procedures (1)(in which a vaporized sample is mixed with a large excess of diluent gas and deposited on a cryogenic 0003-2700/81/0353-1244$01.25/0
surface) are characterized by random orientations of sample molecules if a “conventional”matrix, such as argon or nitrogen, is employed (10). Hence, this paper will not consider sample preparation techniques which produce oriented solute molecules. The results of this paper will therefore be applicable to samples which are isolated in “conventional” matrices or frozen in low-temperatureglasses but will not necessarily apply to samples prepared in crystalline hydrocarbons, Shpol’skii frozen solutions, or mixed crystals.
THEORY In order for a radiation-induced transition to be observed between initial and final states with wave functions \ki and \kf, respectively, the electric dipole transition moment = J.IriF.Irf
a7
must have a nonvanishing value, because the Rrobability for a transition is proportional to the square of Mi,f (9, 11-13). Here, r‘ is the electric dipole vector and r symbolizes the set of internal coordinates Fquired for locating each electron of the molecule. Because Mi$is a vector with components along the molecule-fixed x , y, and z axes, the integral in eq 1 can often be separated into three independent integrals of the form (12, 13) &i,f = f S \ k i z Y f dr J?S\kiyQf d7 + RJQizqf dr (2)
+
where l, j , and ff are unit vectors directed along the x , y, and z molecular axes, respectively. Thus, a transition is electric-dipole-allowed if one of the three integrals in eq 2 does not vanish, which can be determined by group theoretical analysis if the symmetry of the wave functions is known (9, 12, 13). Transitions are generally polarized in the sense that the three integrals in eq 2 are not necessarily equal (12). For example, if the first integral has a finite value while the other two are equal to zero, the transition is said to be polarized along the x molecular axis (9,12,13). In some cases for which the molecular symmetry is sufficiently high or low and where x and y jointly form the basis for an irreducible representation, the integrals involving x and y are not independent of each other and neither integral is equal to zero (12,13). For this situation, the transition is said to be polarized in the xy plane. Of particular interest is the orientation of the transition moment relative to a molecule-fixed coordinate system. In general, the orientation cannot be determined from group 0 1981 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 53, NO. 8, JULY 1981 a A
-C
’t
&>:*
[Clf+
Lb
i
Bb
f
both directions a and c for each example since molecules with other orientations will absorb, but with smaller probabilities. However, the relative degree of the polarization of fluorescence which is observed (in either direction a or c) will be different in these two examples. This is because the polarization of fluorescence (observed in each direction) depends on the relative orientations of the transition moments for absorption and emission, as will now be demonstrated in a quantitative form. For randomly oriented molecules, the intensities of fluorescencepolarized along each of the laboratory-fixed axis a, b, and c in Figure 1 are respectively given (excluding irrelevant proportionality factors) by I, = p [ q x ( 3 r x+ ry) + qy(rx+ 3ry)l + (1- p ) I b ( 3 ) Ib
\Lb
Pyrene
Chrysene
Benzlaianthracene
Flgure 1. The geometry of tluorescence photoselection experiments Is depicted in (a)and (b), and some transition moments for (c)pyrene, (d) chrysene, and (e) benz[ alanthracene are labeled.
theoretical considerations alone. For example, consider the benz[a]anthracene (BaA) molecule, which has C, symmetry (see Figure le). Although transitions which are polarized along the z axis (Le,, perpendicular to the molecular plane) are electric dipole allowed, v i r * transitions with this polarization have not been observed for BaA or any other PAH or derivative (14-18). Consequently, all observed transitions for BaA are between states which belong to the A‘ representation (14). Because x and y both belong to the A’ representation, all transitions in BaA are allowed. However, it should be noted that the transitions are not ( x y ) plane polarized since x and y do not jointly form the basis for an irreducible representation. Instead, the transitions are polarized in particular directions in the xy plane. Furthermore, the polarization of each transition will be different because of the dependence of the transition moment on the particular states which are involved in the transition. For example, Figure 1 illustrates the transition moment orientations for a few transitions in pyrene (P), chrysene (C), and BaA. These orientations are known from experiment in the case of pyrene (14, 19) and chrysene (17) or from free-electron theory in the case of chrysene and BaA (19). In all cases these transitions are between the totally symmetric ground state and an excited singlet state. Hence, the transition moments are labeled by a notation (14,15, 19-21) which signifies the identity of the excited state (i.e., ‘La, ‘Lb, The method of polarized emission and absorption, as applied to randomly oriented molecules, was developed by Albrecht and has been called “the method of photoselection” (9,11). In order to illustrate the concept of photoselection, consider the excitation of the ‘Lb electronic state of pyrene molecules with 370-nm radiation incident along direction b and polarized in the ab plane, as in Figure 1. Since the probability for absorption (or emission) is proportional to c q 2 P, where P is the angle be tween the electric field vector (E) of the incident radiation and the transition moment vector, molecule 1 will absorb and molecule 2 will not. That is, molecule 1is photoselected. Since pyrene is known to always emit x-axis polarized radiation from the lLh electronic state (14, 19), the fluorescence from molecule 1 can be observed along direction c but not a. If the photoselecting light in the example just discussed has a wavelength of ca. 335 nni, corresponding to the excitation of the ‘La state, then pyrene will absorb along they molecular axis (14, 19). That is, molecule 2 will absorb, but molecule 1will not. In this case the fluorescence from pyrene molecule 2 will still be x polarized and can be viewed from direction a, but not c . In reality, fluorescence can be observed from
1245
= qx(rx+ 2ry) + qy(2rx + rY)
I, = (1 - p ) [ q , ( 3 r , + ry) + qY(rz + 3rJl
(4)
+plb
(5)
when the photoselecting light is incident along b and polarized with fraction p along a and ( 1 - p ) along c (9, 11). Here, r, and ry are the probability at a given wavelength for a photon to be absorbed along the x and y molecular axis, respectively (r, + ry = 1). Likewise, the probability for fluorescence (once absorption has occurred) along the x and y axes is denoted by q x and qy, respectively. For simplicity, a fluorescence quantum yield of unity is assumed (i.e., q, + qy = 1). In eq 3-5 it has been assumed that there is no probability for a radiation-induced transition along the z molecular axis, which is perpendicular to the molecular plane (Le., r, = qr = 0). For the planar PAHs this assumption is justified on theoretical grounds (16, 19,20) and by experiment (14-18) for most, if not all, known cases of singlet-singlet absorption and fluorescence. For convenience, eq 3-5 can be reformulated in terms of the angle 0 between the transition moments for absorption and emission. By arbitrarily defining the x axis as the axis along which the fluorescence occurs (i.e., q x = 1, qy = 0),the probabilities for absorption along the x and y axes are r, = cos2 0 and ry = sin2 8. Substituting in eq 3-5 we have
+ sin2 8) + (1- p ) l b I b = cos2 8 + 2 sin2 0 I, = (1- p ) ( 3 cos2 8 + sin2 0) + p l b
I, = p ( 3 cos2 8
(6) (7) (8)
With a knowledge of the angle 0 between the absorption and emission electronic transition moments for the molecule of interest, eq 6-8 can be used to predict the relative polarization of the fluorescence (e.g., I a / I b ) . For example, with either 273 nm (‘Bb) or 370 nm (%b) excitation of pyrene, the absorption is known to occur along the x (short) axis (14,19). Since the fluorescence of pyrene originates from the lowest excited singlet state (SI*, 370 nm), the fluorescence will always be polarized along the short axis. Thus, for photoselecting light incident in direction b and polarized along direction a (i.e., p = l ) ,the theoretically expected intensity of fluorescence polarized parallel to the excitation (along direction a) relative to the perpendicular component of intensity is I a / I b = 3 / 1 = 3. In practice, the theoretically expected polarized intensity ratios in eq 6-8 are virtually never realized (9, 11, 16, 18). Explanations which have been offered for the discrepancies include the depolarizing influence of strains in rigid matrices (16) and polarization anomalies introduced by the optical instrumentation used for the measurement (18),such as the grating in a monochromator. Local heating effects in lowtemperature matrices, which result from radiationless energy transfer processes and which can allow reorientation of the molecules, have also been suggested as a source of discrepancy (9).
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 8, JULY 1981
As another example, the fluorescence of chrysene is known to occur from the ‘Lb electronic state, which is polarized as diagrammed in Figure 1(17,19). Because of the low symmetry (C,) of chrysene, the transition moments cannot be readily deduced and there is disagreement in the literature concerning the assignments for some transitions. For example, with respect to the ‘Lb transition Becker et al. assigned the IBb transition (260-280 nm) to have a parallel polarization (14), while others have found evidence for this transition moment to be directed a t a 60’ angle (in the plane of the chrysene molecule) (17,19). In the former case, 0 = Oo and the predicted relative intensities of fluorescence polarized parallel (I,)and perpendicular (Ib) to an excitation source directed along direction b and polarized along direction a are 3 and 1, respectively (Le., the same as in the previous example with pyrene). In the latter case, 0 = 60’ so that I, = 1.50 and I b = 1.75. In order to predict the analytical utility of fluorescence photoselection, we should determine the maximum possible spectral discrimination which can be achieved for two compounds A and B whose spectral features overlap. The maximum effect can easily be shown to occur when the angle 0 between the emission and absorption transition moments is 8~ = 0’ (for compound A) and OB = 90’ (for compound B). In this case, assuming p = 1in eq 2, I/ = 3.0, I b A = 1.0,I,B = 1.0, and IbB = 2.0. Thus, for a polarizer oriented parallel to the photoselecting light, a factor of I,“/I: = 3.011.0 = 3.0 change in the relative peak heights of A and B would result, while for a perpendicular orientation, IbA/IbB = 1.0/2.0 = 0.50. That is, the relative peak heights for compounds A and B can theoretically vary by a factor of as much as 3.010.5 = 6.0, depending on the orientation of the polarizer with respect to the polarization of the exciting light. The actual spectral enhancement may be somewhat smaller because of the depolarizing effects already discussed. That the relative fluorescence intensities of two compounds can be varied by at most a factor of 6 places a self-evident restriction on analytical applications of the fluorescence photoselection technique. If the fluorescence intensity of an interferent grossly exceeds that of the analyte, any variation of the relative intensities by fluorescence photoselection would be negligible and there would be little advantage in using this technique. Of course, other common procedures, such as selective excitation, generally also fail in such circumstances. Nevertheless, if the anal@ and interferent exhibit comparable fluorescence intensities, the photoselection technique should be useful for confirmation of a tentative identification of the analyte and/or to selectively mask the fluorescence of the interferent to allow a more reliable quantitative determination of the analyte. An additional phenomenon that should be noted is that the observed polarization of fluorescence from a molecule often exhibits a dependence on the vibrational states involved in the transition (9,16-18). The reason is that the vibrational motion can change or destroy the symmetry of a molecule, causing a dependence of the transition moment on the initial and final vibrational states of the transition (9, 16). One consequence of this phenomenon is that the fluorescence spectrum of a particular compound may depend on the orientation of a polarizer used for selectivelypassing fluorescence of a given polarization. In addition, the relative degree of polarization of fluorescence will often vary throughout an absorption band (16-18), due to excitation of different vibrational states of a given electronic band as a function of wavelength. EXPERIMENTAL SECTION Commercially available “research grade” nitrogen gas and perfluoro-n-hexane (PCR,assay 99% perfluorohexanes, 85% “n-”)
Figure 2. Dye laser Induced (270 nm) fluorescence spectra of 80 ng pyrene matrix isolated In nbogen, whlle using (a) no polarizer and whlle using a polarizer oriented (b) perpendlcular and (c) parallel to the polarizatlon of the laser pulses. In cases (b) and (c), the polarlzer was situated between the sample and the emission monochromator.
were used as matrix materials without further purification. The 1,7-dihydroxynaphthalene(Aldrich) was vacuum sublimed twice prior to use. All samples were deposited on a gold-plated copper surface, which was maintained at 15 K in the head of a closed-cycle helium cryostat (“Spectrim”, CTI-Cryogenics, Waltham, MA). A Knudsen effusion, vacuum sublimation apparatus which has been previously described was used for depositing the samples (I,22). A Molectron DL-14 dye laser, pumped by a Molectron UV-24 nitrogen laser, was used as the polarized excitation source for these experiments. In normal operation the dye laser produced pulses (6 ns fwhm, 0.02 nm bandwidth, and a pulse repetition rate of ca. 10 Hz) which were directed along b in Figure 1and polarized along a, so that p = 1in eq 1and 2. Dye laser output at 270 nm was achieved with M proprietary fluorinated coumarin (C495) dye (Molectron Corp., Sunnyvale, CA), followed by frequency doubling with an angle-tuned ADP crystal. Polarized fluorescence spectra were obtained by filtering the sample fluorescence (emanating in direction c in Figure 1)with an HNP’B (Polaroid) polarizer, oriented either parallel or perpendicular to the laser polarization direction. The fluorescence was then dispersed by a 1-mgrating monochromator (Jobin-Yvon HR-1000, equipped with an 1800 groovelmm holographic grating blazed at 300 nm), which exhibited a reciprocal linear dispersion of 0.8 nmfmm, and was detected with an RCA 8850 photomultiplier. The photomultiplier signal was fed to a Tektronix 5440 oscilloscope, equipped with a 5S14N dual-trace delayed-sweep sampling plug-in (rise time = 350 ps), prior to plotting by an X-Y recorder. Although it is likely that our monochromator exhibits a polarization-dependent efficiency, our results depend only on relative intensity changes, and this experimental artifact is consequently unimportant. RESULTS AND DISCUSSION Figure 2 compares the fluorescence spectra of pyrene (matrix isolated in nitrogen) with no polarizer and with a polarizer oriented perpendicular and parallel, respectively, to the exciting light. Each peak in these spectra arises from a transition from the ground vibrational state of SI*to a different vibrational level of the ground singlet state. As noted above (cf. Theory), the relative intensities of the various vibronic bands are expected to vary with the orientation of the polarizer. Effects such as those depicted in Figure 2 are reproducible and can serve as an additional parameter for confirming identifications of specific compounds in multicomponent samples. As an example of how the phenomenon of polarized emission can be used to alter the relative contributions of individual PAHs to the fluorescence spectrum of a mixture, Figure 3 shows dye laser-induced fluorescence spectra of a simple mixture of pyrene and chrysene. The polarized 270-nm excitation is absorbed by the ‘Bb band for both compounds (14,17,19). The middle and right spectra were obtained with a polarizer oriented perpendicular and parallel, respectively, to the excitation polarization. Obviously, the middle spectrum exhibits enhanced spectral discrimination of chrysene, while, in the right spectrum, the spectral features of pyrene are enhanced. This example was chosen to illustrate the phe-
ANALYTICAL CHEMISTRY, VOL. 53, NO. 8, JULY 1981
1247
I I
Figure 3. Dye laser Induced (270 nrn) fluorescence spectra of 100 ng each of pyrene (P) and chrysene (C) matrix isolated in nitrogen, while using (a) no polarizer and whlle using a polarizes oriented ( b ) perpendicular and (c) parallel to the polarization of the laser pulses.
nomenon of photoselection in a mixture; in practice (as discussed below), practical analytical applications of photoselection usually will involve samples for which major band overlaps cannot be avoided. The modification of the laser spectra by a polarizer indicates that the polarization assignment made by Becker et al. (14) of the 'Bb absorption band for chrysene is in error. That is, based on their assignment, the theoretically expected ratio of I a / I b = 3 is the same as for pyrene, indicating that no polarization effects should be observed for the spectra in Figure 3. It is instructive to determine whether the polarization assignment for the 'Bb absorption band of chrysene given by the experimental and theoretical results of Gallivan and Brinen (17)and of Ham and Ruedenberg (19), respectively, is sufficient to predict the results shown in Figure 3. As noted above, the relative intensities of fluorescence polarized perpendicular (Ib) and parallel (I,)to the excitation polarization are respectively (in this case) 1.0 and 3.0 for pyrene and, for chrysene, 1.75 and 1.50. Thus, by orienting the polarizer perpendicular to the excitation polarization, a reduction in the fluorescence intensities (or peak heights) of pyrene (P) relative to chrysene ( C ) should be I b p / I b c = 1.0/1.75 = 0.57. By measuring the peak heights in Figure 3 (left and middle) of the 0-0 emission bands for chrysene at 358 nm and for pyrene a t 370 nm, we obtain a reduction in relative intensities of 0.64. Likewise, the theoretically predicted value for the increased intensity of pyrene relative to chrysene fluorescence in Figure 3 (right) is IaP/I: = 3.0J1.50 = 2.00, while our measurement yields the value of 1.75. The agreement of our results with those of Ham and Ruendenberg (19) is quite good, especially considering that no attempt was made to correct for the spurious depolarizing effects mentioned above. Another reason for the discrepancy between our results and those of Ham and Ruendenberg is due to the fact that excitation at 270 nm probably does not correspond to the purely electronic excitation of the 'Bb excited states of either pyrene or chrysene (Le., without concomitant vibrational excitation). A more analytically realistic example of fluorescence photoselection in low-temperature matrices is shown in Figure 4. Laser pulses a t 270 nm were used to excite a mixture of 57 ng of pyrene and 100 ng of BaA isolated in a nitrogen matrix; serious overlaps between major spectral features of these compounds are noted (Figure 4, left). In the center spectrum in Figure 4, there is a considerable enhancement of the spectral features of the carcinogenic BaA when a polarizer oriented perpendicular to the exciting light is used. In this case it would obviously be difficult to positively identify BaA without the use of a polarizer. For BaA the 270-nm excitation apparently induces the 'Bb transition, whose (purely electronic) origin has been reported at 288 nm (14). With this assumption the angle 6 between the 'Bb absorption and 'Lb emission transition moments, which are illustrated in Figure 1, is 80' (19). From eq 6 and 7, I, = 1.06 and I b = 1.97.
Figure 4. Dye laser
induced (270 nrn) fluorescence spectra of 100
ng of benz[a]anthracene (BaA) and 57 ng of pyrene (P) matrix Isolated in nitrogen while using (a) no polarizer and while uslng a polarizer oriented (b) perpendicular and (c)parallel to the polarization of the laser
pulses. Therefore, by orienting the polarizer perpendicular to the exciting light, an increase in the intensity of fluorescence of BaA to pyrene should be IbBd/IbP = 1.97J1.0 = 1.97, while a parallel orientation of the polarizer should result in a IaB*/I,P= 1.06J3.0 = 0.35 decrease in the emission of BaA relative to pyrene. By use of the 370 nm and 381 nm peak heights for pyrene and BaA, respectively, our measurement results in changes in the intensity ratios of BaA with respect to pyrene of 2.46 and 0.66 for a polarizer oriented perpendicular and parallel, respectively, to the polarization of the laser light. The differences between these measurements and the theoretically expected ratios are quite large, presumably because of the severe overlap of the 381-nm BaA peak with the vibronic bands of pyrene. In order to test this explanation, we deposited a smaller amount (12 ng) of pyrene in a nitrogen matrix with 100 ng of BaA. The resulting changes in the intensity ratios of BaA with respect to pyrene were 1.90 and 0.59, respectively, for a polarizer oriented perpendicular and parallel to the exciting light. The differences between these values and the previously computed theoretical values of 1.97 and 0.35 are significantly closer than the values obtained from the deposit with 57 ng of pyrene, indicating that the discrepancies may be due (to a large extent) to the spectral overlap of pyrene and BaA peaks. This experiment also seems to confirm that the 270-nm excitation of BaA is indeed in the 'Bb absorption band, instead of one of the nine other known electronic absorption bands of BaA (14). The fluorescence photoselection technique can also be extended to exploit vibrational polarization effects. As discussed above, the direction of the transition moment for absorption or emission often depends on the symmetry of the vibrational modes involved in the transition (Figure 2). This is especially true for electronic transitions which are weakly allowed, in which case the direction of the transition moment may be predominately dependent on the symmetry of the vibrational modes (9, 16). The most promising applications of the vibrational mode dependence of the transition moment probably will employ fluorescence photoselection in conjunction with site selection or selective excitation techniques for the analysis of PAH derivatives. The primary difficulty in characterizing a complex mixture of derivatives of any particular PAH is that the absorption and emission bands of the derivatives are generally quite similar to those of the parent PAH (5,151,resulting in considerable overlap of both absorption and emission bands. This problem can be dealt with, in principle, by excitation into the lowest energy excited electronic state, because the vibronic bands of this state are usually resolved to a much greater extent than the bands due to higher energy electronic states (2,5, 6, 15,161,thus presenting significantly greater possibilities for the selective excitation of a component of interest in the mixture (2, 5). However, depending on the
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 8, JULY 1981
330
390
330
390
nm
Figure 5. Dye laser induced (327.9 nm) fluorescence spectra of 150 ng of 1,7dihydroxynaphthalenematrix Isolated in perfluoro-n-hexane while using (left)no polarizer and while using (right) a polarizer oriented
perpendicular to the polarization of the laser pulses.
complexity of the sample, the overlap of vibronic bands of different PAH derivatives may still be appreciable. Because the lowest energy excited singlet state of PAHs and derivatives is often only weakly allowed (14-16), the transition moment for absorption and emission will be strongly influenced by the symmetry of the vibrational modes which are involved in the transition. Thus, the method of fluorescence photoselection may be expected to be helpful in resolving overlapping fluorescence bands in this situation. This technique has been applied indirectly to the analysis of a complex mixture of naphthalene hydroxyl derivatives (5). The laser-induced fluorescence spectra in Figure 5 of 1,7dihydroxynaphthalene (1,7-DHN) matrix isolated in perfluoro-n-hexane illustrate the potential of fluorescence photoselection for the resolution of mixtures of naphthalene derivatives. The spectrum on the left in Figure 5 was obtained without a polarizer, while a polarizer oriented perpendicular to the polarization of the laser output was used for the spectrum on the right. In order to account for the loss of intensities of the long wavelength emission bands relative to the 0-0 band in the spectrum on the right, it is postulated that the transition moment of the particular vibronic band which has been excited is polarized perpendicular to the transition moment of the purely electronic (0-0) emission band and is polarized parallel to the transition moments of many of the longer wavelength emission bands. Because of the relative loss of intensity of the higher wavelength vibronic emission bands, the intensity of the broad phonon wings (5) which accompany each vibronic band are also reduced, resulting in a considerable improvement in the base line resolution of 1,7-DHN and in an increased probability of detecting other components which fluoresce at longer wavelengths than the 0-0 emission band of 1,7-DHN (332.7 nm). An important advantage of the fluorescence photoselection methodology is that it can easily be applied in conjunction with selective excitation (2-4), site selection (5-7), and time resolution (7,8)low-temperature fluorometric methods which have already been developed for alleviating the problem of spectral overlap. However, situations may often be encountered where selective excitation and site selection techniques, which usually require excitation into the lowest energy singlet absorption band (2,5, 7),may not be advantageous for real sample analysis, especially when the analyte is present at very low concentration. In these situations, in which detectivity is the primary analytical challenge, excitation into higher energy absorption bands is preferred, since the molar extinction coefficients are generally much larger than for the lowest energy (singlet)absorption band (14,15). For instance, the 'Lb and 'Bb transitions of BaA are characterized by molar extinction coefficients of 850 and 95000, respectively (14).
Although frequency doubling of the dye laser output is required for excitation into the 'Bb absorption band (270-295 nm) of BaA, resulting in a large (ca. 90%)loss of peak power, the detectivity for 'Bb excitation should exceed that for 'Lb excitation by a factor of 10. Thus, in this case ('Bb excitation) fluorescence photoselection would prove especially useful as a method for increasing the selectivity of the analysis if pyrene (a common interference in low-temperature fluorometry) were also present in the sample. Finally, the importance of fluorescence photoselection as an analytical procedure will be particularly noteworthy in situations where site selection and selective excitation procedures cannot be applied at all, such as for sterically hindered molecules such as benzo[c]phenanthrene whose absorption and fluorescence bands are characterized by severe homogeneous broadening. Another common situation is for compounds whose lowest energy vibronic absorption bands are inaccessible to the available excitation source. For example, because of a gap from 330 to 363 nm in which we have been unable to obtain useful output from our dye laser, these more sophisticated procedures cannot be utilized for determinations of many three- or four-ring heterocycles and PAHs (e.g., chrysene and BaA). In these situations fluorescence photoselection and time resolution are virtually the only fluorometric methods which can be employed to enhance the analytical selectivity. Obviously, the selectivity will depend exclusively on the excitation wavelength and on the properties and quantity of the interferences. The examples used in this paper were selected because the polarizations of the electronic transitions were documented in the literature. In general, however, the polarization of many purely electronic transitions in two- to four-ring PAHs is not known or else there are extensive contradictionsor ambiguities concerning the assignments in the literature (14-18). Even less is known about the polarizations of vibronic transitions, while for most five-ring or larger PAHs, there is absolutely no information in the literature concerning the polarization of the transitions (14-18). Nevertheless, the method of fluorescence photoselection can be applied to any occurrence of spectral overlap in a multicomponent sample by orienting a polarizer both parallel and perpendicular to the polarization of the photoselecting light. This empirical application of the technique is sufficient for quantitative applications because it can be shown that the maximum change in relative intensities for the overlapping bands will always occur for these two specific orientations of the polarizer. For qualitative analysis the expected relative intensity changes for the analyte and interference may need to be known before the analyte can be positively determined. An exception to this restraint is for situations in which more than one band from the anal@ can be observed by using fluorescence photoselection. LITERATURE CITED (1) Wehry, E. L.; Mamantov, 0.Anal. Chem. 1979, 51, 643A-654A. (2) Maple, J. R.; Wehry, E. L.; Mamantov, G. Anal. Chem. 1980, 52, 920-924. (3) Wehry, E. L.; Mamantov, G.; Hembree, D. M.; Maple, J. R. In "Polynuclear Aromatic Hydrocarbons: Chemistry and Blologlcai Effects"; Blorseth, A., Dennis, A. J., Eds.; Battelle Press: Columbus, OH, 1980; p 1005. (4) Yang, Y.; D'Silva, A. P.; Fassel, V. A.; Iles, M. Anal. Chem. 1980, 52, 1350-1351. (5) Maple, J. R.; Wehry, E. L. Anal. Chem. 1981, 53, 266-271. (6) Brown, J. C.; Edelson, M. C.; Small, 0. J. Anal. Chem. 1978, 50, 1394-1397. (7) Brown, J. C.; Duncanson, J. A,, Jr.; Small, G. J. Anal. Chem. 1980, 52, 1711-1715. ( 8 ) Dicklnson, R. B., Jr.; Wehry, E. L. Anal. Chem. 1979, 51, 778-780. (9) Albrecht, A. C. J . Mol. Specfrosc. 1981, 6 , 84-106. (10) Meyer, B. "Low Temperature Spectroscopy"; American Elsevler: New York, 1971; p 310. (11) Albrecht, A. C.; Simpson, W. T. J . Am. Chem. SOC. 1955, 77, 4454-4461. (12) Hochstrasser, R. M. "Molecular Aspects of Symmetry"; W. A. Benjamin: New York, 1966; pp 215-219.
Anal. Chem. 1981, 53, 1249-1253 (13) Cotton, F. A. “Chemical Applications of Group Theory”; Wiley-Interscience: New York, 1963; pp 102-104. (14) Becker, R. S.; Singh, I.S.; Jackson, E. A. J . Chem. Phys. 1963, 38, 2144-21 7 I . (15) iaffd, H . H.; Orchin, M. “Theory and Applications of Ultraviolet SDectroscoDv”: Wiiev: New York. 1962: DD 294-344. (16) Murreil, J. ‘N. ‘“The-Theory of the Elect;onlc Spectra of Organic Molecules”; Why: New York, 1963; pp 91-132. (17) Gailivan, J. 8.; Brlnen, J. S. I n “Molecular Luminescence”; Lim, E. L., Ed.; W. A. Benjamin: New York, 1969; pp 93-110. (18) Azumi, T.; McGlynn, S. P. J. Chem. Phys. 1962, 37, 2413-2420. (19) Ham, N. S.;Ruedenberg, K. J . Chem. Phys. 1956, 25, 13-26.
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RECEIVED for review January 5,1981. Accepted April 15,1981. This research was supported in part by National Science Foundation Research Grants CHE77-12542and cHE80-25282 to the University of Tennessee.
Determination of Trace Element Profiles and Concentrations in Human Hair by Proton-Induced X-ray Emission Spectrometry John L. Campbell,” Shatha Falq, Rosalind S. Gibson,’ Sean B. Russell, and Christian W. Schulte Department of Physics, University of Guelph, Guelph, Ontario, Canada N 1G 2 W I
The effect of proton bombardment on the integrity of human hairs is studied via Rutherford scattered protons and emltted characteristic X-rays. At the current densities necessary for rapid PIXE analysis, damage can be minimized by use of a helium atmosphere. The scanning of individual hairs with about 1 mm resbiution and the subsequent conversion of one hair to a thin target are described. The first measurement provides a position dependence of trace element concentrations in relative terms; the second provides absolute concentrations for the entire hair. The position-dependent data can then be converted into concentrations. This provides a history of trace element deposition into the growing hair over a period of several moriths with a time resolution of a few days. An application to druginduced zinc deficiency in human subjects is described.
A human hair consists of three main components as depicted schematically in Figure l. The hard outer cuticle is a layer of scales encasing and protecting the cortex, which in turn is a stable fibrous protein (keratin). In the center of the cortex is a continuous or discontinuous medulla, composed of large cornified cells, loosely linked together; in fine hairs this is often absent. Trace elements are incorporated into the proximal end of a growing hair BS it emerges (at 0.2-0.5 mm per day) from the follicle. Hopps (1) lists the endogenous sources of trace elements in scalp hairs as: (i) the matrix and connective tissue papilla with its blood and lymph vessels (major); (ii) the sebaceous glands in the skin which provide trace elements from body tissues (minor); (iii) the eccrine sweat glands which also provide trace elements from body tissues (minor); (iv) the epidermis (minor). One therefore expects the changes in trace element content along the hair shaft to reflect to some extent the varying trace metal status of the subject; this makes hair a unique biopsy material. Trace element analysis of hair has long been a contentious topic, and many attempts to correlate the trace element content of bulk hair samples with the content of other specific tissues have yielded disappointing results. Part of the problem undoubtedly lies in hair’s susceptibility to exogenous contamination from the atmospheric aerosol and from cosmetics; there are also large variations with age, sex, geography, diet 0003-2700/81/0353-1249$01.25/0
etc. Valkovii: (2) expresses the opinion that poor sampling, improper preparation, and inappropriate statistical analysis are easily recognized in the majority of papers on hair analysis. Nonetheless Hopps (1)finds strong support from a large body of literature for the view that the trace element content of hair does reflect the overall body intake and hence the trace element status. For example, specific well-controlled studies have recently demonstrated strong relationships between changes in trace elements in hair, serum, and the diet during early infancy (3, 4). Most analyses of hair have used either neutron activation or atomic absorption, both destructive techniques generally requiring a minimum of several strands of hair. It is possible with NAA to cut one hair in segments and analyze these to obtain a trace element profile along the hair, but as the segment size approaches a few days’ growth (1-2 mm) sensitivity is lost. The newer technique of proton-induced X-ray emission (PIXE) is nondestructive, and since proton beams can be focussed to 10 pm diameter, it permits longitudinal scanning with a (possible) effective time resolution (in terms of hair growth) of a few hours. Given the mixed record of bulk hair analysis to date, one must ask if elemental profiles along an individual’s hair may be more rewarding in terms of biomedical information than comparison of bulk concentrations among individuals and controls. Two brief early papers demonstrated the potential of PIXE for such profiling. Valkovii: et al. (5) cut single hairs into 14 segments each of 3 cm length and bombarded these with 3-MeV protons; the resulting X-ray spectra showed that elemental X-ray intensities varied markedly along the hairs. Horowitz and Grodzins (6)used a proton microbeam of 25 hm diameter to scan along the hair of an individual who had ingested methylmercury; the mercury profile clearly reflected this event. Workers subsequently applying PIXE to hair analysis have tended to focus on environmental pollution. For example Rendic et al. (7) demonstrated the absorption by hair of the air pollutants lead, bromine, arsenic, and strontium by measuring the X-ray intensities of these elements relative to that of zinc; a monotonic increase in the ratios with distance from the scalp was observed. The nuclear microprobe group (8) at UKAEA’s Harwell Laboratory has performed a large number of hair analyses; one example (9) was concerned with possible ingestion of arsenic after an accident. Henley et al. 0 1981 American Chemical Society
