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Comment on laser-excited fluorescence line-narrowing: An analytical study
Tolanta, Vol. 31, No. 9, pp. 741-742, Printed in Great Britain
0039-9140/84 $3.00 + 0.00 Pergamon Prss Ltd
1984
ANNOTATIONS COMMENT ON LASER-EXCITED FLUORESCENCE LINE-NARROWING: AN ANALYTICAL STUDY J. M. HAYESand G. J. SMALL Ames Laboratory-USDOE, Iowa State University, Ames, Iowa (Received I December
50011,
U.S.A.
1983. Accepted 20 April 1984)
Summary-A recent paper (D. Bolton and J. D. Winefordner, Talunta, 1983,30,9) on the analytical utility of laser-excited fluorescence line-narrowing is critically assessed.
In a recent paper’ Bolton and Winefordner (BW) reported on the limit of detection (LOD) for fluorescence line-narrowing spectroscopy (FLNS) of polycyclic aromatic hydrocarbons (PAHs) in a glycerol:ethanol:water glass at 4.2 K. The PAHs studied were anthracene, 2- and 9_methylanthracene, 9,1 Odimethylanthracene, perylene and pyrene. The LOD values were compared with those determined for the same compounds by room-temperature fluorescence and low-temperature fluorescence spectrometry. In our opinion the BW paper presents a very misleading assessment of the analytical utility of FLNS. Because of our experience in exploring this question2-9 we feel compelled to make the following comments. 1. The procedure and apparatus used by BW for FLNS are similar to those employed by us. The liquid-helium bath, sample preparation, sample cooldown procedures and monochromator bandpass (1 A) are identical. Comparison of the N,-pumped dye lasers, monochromator apertures and slit-widths utilized by BW and by us indicates there should be approximate parity in sensitivity. Nevertheless, for the compounds listed, BW report LOD values between 100 and 1000 ng/ml. For the same compounds and several others our standard FLN spectra were generally obtained with very high signal-to-noise ratio (S/N) at 200 ng/ml.3,5 Indeed, for perylene (BW LOD = 100 ng/ml), pyrene (BW LOD = 300 ng/ml), and 1-methylpyrene we obtained a detection limit (S/N > 5) of -0.02 ng/ml.5 In the direct analysis of a solvent-refined coal II sample, I-methylpyrene, benzo[e]pyrene, benzo[u]pyrene, anthracene, perylene and benzo[k]fluoranthene were characterized and determined at concentrations ranging between -2 and 30 ng/ml (in the glass).’ Thus, the rather bleak sensitivity picture for FLNS painted by BW is hardly warranted. As discussed elsewhere,3,5 with improvements in the FLNS apparatus used in reference 3, detection limits of 1 pg/ml are readily
achievable for strongly fluorescent PAHs in pure solvents. 2. The failure of BW to achieve with FLNS the respectable LOD for PAHs that is routinely obtained in our laboratory may be due, in part, to the different gated-detection electronics and datahandling procedure they utilize. They mention a problem with high background signals but are not specific about it. We have never experienced background interference except at very low concentrations (4 1 ng/ml).’ Nor has small bubble formation from boiling He posed an insurmountable problem as it did in their w0rk.j However, another problem in the BW studies appears to be their choice of excitation wavelength (Iz,,) and emission wavelength (&,,). For example, in the case of anthracene, A,, = 364.0 nm (as used by BW) excites vibronic bands at an energy level some 1200 cm-’ above the S, origin. At this energy there is considerable overlapping of vibronic bands, so FLN is observed from several isochromats. Although a similar 1,, (363.8 nm) was used in reference 2, that work was done with a non-tunable Ar+ laser. A better choice of de,,is given in references 3 and 5. In that work & = 373.8 nm was used. This corresponds to excitation into the centre of the first vibronic band, so that only a single isochromat will fluoresce. Consider also the case of 9-methylanthracene. The I,, = 382.5 nm chosen by BW is midway between the centres of two overlapping vibronic absorption bands. Thus two bands, separated by the vibronic energy difference, will emit. This emission will be weak because the excitation is into the tail of each band. Furthermore, the most intense zero-phonon line associated with the emissions will be at - 388 nm and - 393 nm, respectively. The I,, value chosen by BW, however, is 390.6 nm, in a “valley” nearly midway between the two zerophonon lines! Finally, it has been observed in our laboratory that 2-methylanthracene is not amenable to FLN analysis in glycerol, water, ethanol glasses at concentrations greater than 100 ng/ml (BW’s LOD
741
142
ANNOTATIONS
for this species). At these concentrations the emission is broad, owing to aggregation.’ 3. BW assert that the loss of FLN behaviour when lc2,, pumps uossessing
vibronic levels of the fluorescent state 2 1500 cm-’ excess vibrational energy is
due to “rapid local site-melting”. This explanation is erroneous. The correct explanation2~3~‘o has to do with the fact that at such high excess vibrational energy several isochromats belonging to different overlapping vibronic absorption bands are pumped. In conclusion, BW report LOD values for FLNS which are anomalously high, do not discuss their results in the light of those from earlier work,3,S and ignore several other attributes of the technique. For example, FLNS possesses high selectivity,3-9 which was initially the reason why our laboratory pursued and developed it as an analytical technique. Another example is that water-based glasses impart to it a versatility which other solid-state fluorescence methods do not possess. For example, by acidification of the glass the amino-derivatives of PAHs can be studied.7e9 As another example we cite recent work where we have successfully applied FLNS to aromatic carcinogen-DNA adducts and shown that two adducts formed by different metabolic paths for benzo[a]pyrene can be distinguished in a mixture.‘.”
Acknowledgemenr-Ames Laboratory is operated for the U.S. Department of Energy by Iowa State University under contract No. W-7405-Eng-82. This research was supported by the Office of Health and Environmental Research, Office of Energy Research.
REFERENCES J. D. Winefordner and D. Bolton, Talanta, 1983, 30,9. 2. J. C. Brown, M. C. Edelson and G. J. Small, Anal. Chem., 1978, 50, 1394. 3. J. C. Brown, J. A. Duncanson, Jr. and G. J. Small, ibid., 1980, 52, 1711. 4. J. C. Brown, J. M. Hayes, J. A. Warren and G. J. Small, in Lasers and Chemical Analysis, G. M. Hieftje and F. E. Lvtle teds.). Humana Press, Clifton. NJ, 1981. 5. J. C. Brown, Ph.D. Dissertation, Iowa State University, 1981. 6. J. M. Hayes, I. Chiang, M. J. McGlade, J. A. Warren and G. J. Small, Proc. SPIE-Inr. Sot. Opt. Eng., 1981, No. 286, 117. I. I. Chiang, J. M. Hayes and G. J. Small, Anal. Chem., 1982, 54, 315.
8. I. Chiang, MS. Thesis, Iowa State University, 1981. 9. M. J. McGlade, M.S. Thesis, Iowa State University, 1983. 10. R. I. Personov, in Spectroscopy and Excirafion of CondensedMolecular Svstems. V. M. Aaranovich and R. M. Hochstrasser (eds.j, Chap. 10. NGrth Holland, New York, 1983. Il. V. Heisig, A. M. Jeffrey, M. J. McGlade and G. J. Small, Science, 1984, 223, 289.
[A reply to this comment will be found on p. 753; Ed.]
0039-9140/84 $3.00 + 0.00 Pergamon Prss Ltd
1984
ANNOTATIONS COMMENT ON LASER-EXCITED FLUORESCENCE LINE-NARROWING: AN ANALYTICAL STUDY J. M. HAYESand G. J. SMALL Ames Laboratory-USDOE, Iowa State University, Ames, Iowa (Received I December
50011,
U.S.A.
1983. Accepted 20 April 1984)
Summary-A recent paper (D. Bolton and J. D. Winefordner, Talunta, 1983,30,9) on the analytical utility of laser-excited fluorescence line-narrowing is critically assessed.
In a recent paper’ Bolton and Winefordner (BW) reported on the limit of detection (LOD) for fluorescence line-narrowing spectroscopy (FLNS) of polycyclic aromatic hydrocarbons (PAHs) in a glycerol:ethanol:water glass at 4.2 K. The PAHs studied were anthracene, 2- and 9_methylanthracene, 9,1 Odimethylanthracene, perylene and pyrene. The LOD values were compared with those determined for the same compounds by room-temperature fluorescence and low-temperature fluorescence spectrometry. In our opinion the BW paper presents a very misleading assessment of the analytical utility of FLNS. Because of our experience in exploring this question2-9 we feel compelled to make the following comments. 1. The procedure and apparatus used by BW for FLNS are similar to those employed by us. The liquid-helium bath, sample preparation, sample cooldown procedures and monochromator bandpass (1 A) are identical. Comparison of the N,-pumped dye lasers, monochromator apertures and slit-widths utilized by BW and by us indicates there should be approximate parity in sensitivity. Nevertheless, for the compounds listed, BW report LOD values between 100 and 1000 ng/ml. For the same compounds and several others our standard FLN spectra were generally obtained with very high signal-to-noise ratio (S/N) at 200 ng/ml.3,5 Indeed, for perylene (BW LOD = 100 ng/ml), pyrene (BW LOD = 300 ng/ml), and 1-methylpyrene we obtained a detection limit (S/N > 5) of -0.02 ng/ml.5 In the direct analysis of a solvent-refined coal II sample, I-methylpyrene, benzo[e]pyrene, benzo[u]pyrene, anthracene, perylene and benzo[k]fluoranthene were characterized and determined at concentrations ranging between -2 and 30 ng/ml (in the glass).’ Thus, the rather bleak sensitivity picture for FLNS painted by BW is hardly warranted. As discussed elsewhere,3,5 with improvements in the FLNS apparatus used in reference 3, detection limits of 1 pg/ml are readily
achievable for strongly fluorescent PAHs in pure solvents. 2. The failure of BW to achieve with FLNS the respectable LOD for PAHs that is routinely obtained in our laboratory may be due, in part, to the different gated-detection electronics and datahandling procedure they utilize. They mention a problem with high background signals but are not specific about it. We have never experienced background interference except at very low concentrations (4 1 ng/ml).’ Nor has small bubble formation from boiling He posed an insurmountable problem as it did in their w0rk.j However, another problem in the BW studies appears to be their choice of excitation wavelength (Iz,,) and emission wavelength (&,,). For example, in the case of anthracene, A,, = 364.0 nm (as used by BW) excites vibronic bands at an energy level some 1200 cm-’ above the S, origin. At this energy there is considerable overlapping of vibronic bands, so FLN is observed from several isochromats. Although a similar 1,, (363.8 nm) was used in reference 2, that work was done with a non-tunable Ar+ laser. A better choice of de,,is given in references 3 and 5. In that work & = 373.8 nm was used. This corresponds to excitation into the centre of the first vibronic band, so that only a single isochromat will fluoresce. Consider also the case of 9-methylanthracene. The I,, = 382.5 nm chosen by BW is midway between the centres of two overlapping vibronic absorption bands. Thus two bands, separated by the vibronic energy difference, will emit. This emission will be weak because the excitation is into the tail of each band. Furthermore, the most intense zero-phonon line associated with the emissions will be at - 388 nm and - 393 nm, respectively. The I,, value chosen by BW, however, is 390.6 nm, in a “valley” nearly midway between the two zerophonon lines! Finally, it has been observed in our laboratory that 2-methylanthracene is not amenable to FLN analysis in glycerol, water, ethanol glasses at concentrations greater than 100 ng/ml (BW’s LOD
741
142
ANNOTATIONS
for this species). At these concentrations the emission is broad, owing to aggregation.’ 3. BW assert that the loss of FLN behaviour when lc2,, pumps uossessing
vibronic levels of the fluorescent state 2 1500 cm-’ excess vibrational energy is
due to “rapid local site-melting”. This explanation is erroneous. The correct explanation2~3~‘o has to do with the fact that at such high excess vibrational energy several isochromats belonging to different overlapping vibronic absorption bands are pumped. In conclusion, BW report LOD values for FLNS which are anomalously high, do not discuss their results in the light of those from earlier work,3,S and ignore several other attributes of the technique. For example, FLNS possesses high selectivity,3-9 which was initially the reason why our laboratory pursued and developed it as an analytical technique. Another example is that water-based glasses impart to it a versatility which other solid-state fluorescence methods do not possess. For example, by acidification of the glass the amino-derivatives of PAHs can be studied.7e9 As another example we cite recent work where we have successfully applied FLNS to aromatic carcinogen-DNA adducts and shown that two adducts formed by different metabolic paths for benzo[a]pyrene can be distinguished in a mixture.‘.”
Acknowledgemenr-Ames Laboratory is operated for the U.S. Department of Energy by Iowa State University under contract No. W-7405-Eng-82. This research was supported by the Office of Health and Environmental Research, Office of Energy Research.
REFERENCES J. D. Winefordner and D. Bolton, Talanta, 1983, 30,9. 2. J. C. Brown, M. C. Edelson and G. J. Small, Anal. Chem., 1978, 50, 1394. 3. J. C. Brown, J. A. Duncanson, Jr. and G. J. Small, ibid., 1980, 52, 1711. 4. J. C. Brown, J. M. Hayes, J. A. Warren and G. J. Small, in Lasers and Chemical Analysis, G. M. Hieftje and F. E. Lvtle teds.). Humana Press, Clifton. NJ, 1981. 5. J. C. Brown, Ph.D. Dissertation, Iowa State University, 1981. 6. J. M. Hayes, I. Chiang, M. J. McGlade, J. A. Warren and G. J. Small, Proc. SPIE-Inr. Sot. Opt. Eng., 1981, No. 286, 117. I. I. Chiang, J. M. Hayes and G. J. Small, Anal. Chem., 1982, 54, 315.
8. I. Chiang, MS. Thesis, Iowa State University, 1981. 9. M. J. McGlade, M.S. Thesis, Iowa State University, 1983. 10. R. I. Personov, in Spectroscopy and Excirafion of CondensedMolecular Svstems. V. M. Aaranovich and R. M. Hochstrasser (eds.j, Chap. 10. NGrth Holland, New York, 1983. Il. V. Heisig, A. M. Jeffrey, M. J. McGlade and G. J. Small, Science, 1984, 223, 289.
[A reply to this comment will be found on p. 753; Ed.]