Anal. Chem. 1988, 60, 1622-1623
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CORRESPONDENCE Multicomponent Suppression of Fluorescent Interferants Using Phase-Resolved Raman Spectroscopy Sir: Weak Raman signals are often overwhelmed by intense background luminescence. To complicatematters even more, the problem is amplified because of the extremely large difference between the Raman-scattering and fluorescencecross sections (1). To help circumvent these interference problems, a myriad of suppression techniques have been developed to minimize the effects of background luminescence in Raman spectroscopy (2). One such suppression-based approach is the use of phase resolution. Briefly, in phase resolution, high-frequency (megahertz-gigahertz) sinusoidally modulated light is used to excite the sample. The resulting fluorescence is phase shifted and amplitude demodulated to an extent that depends critically on the fluorescence lifetime (3). However, because the Raman scatter is essentially instantaneous, it is not phase shifted nor demodulated with respect to the exciting sinusoid. Thus, by employing either phase (4) or modulation (3) selective detection, one can easily resolve Raman from fluorescence. Phase-resolved Raman spectracopy BRRS) simply exploits the phase difference between the fluorescence and Raman emission sinusoids to affect a separation of the two phenomena. Specifically, if we consider a two-component system R and F denoting Raman and fluorescence, respectively, the resulting phase-resolvedintensity (Z(b,A)) takes the form (4, 5)
where is the phase angle for the detector (lock-in amplifier), F(A) is the fluorescence spectrum, R(A)is the Raman scatter spectrum, I$F is the phase angle for the fluorescence process, $R is the phase angle for the Raman scatter (usually zero), MF is the demodulation factor for fluorescence, MR is the demodulation factor for Raman (usually l),and me. is the depth of modulation for the excitation sinusoid. In order to successfully suppress the fluorescence contribution to the total signal, one simply adjusts the detector phase angle such that it is k9Oo out of phase with the fluorescence contribution. The result is Clearly, the remaining h a n scatter is attenuated to a degree dependent on the difference between the relative phase angles of Raman to fluorescence. Maximum enhancement is of course achieved when the fluorescence is phase-shifted 90" with respect to Raman. Figure 1shows the theoretical effect of varying the modulation frequency on eq 2. Specifically, fluorescence lifetimes of 100 ps, 1ns, 5 ns, and 10 11s are shown. The results in Figure 1 are shown as I($D,A) vs modulation frequency (megahertz) and are normalized to the 10-ns value at 275 MHz. The 275-MHz frequency was chosen because it is very near the upper frequency limit for our instrumentation. Importantly, with instrumentation like that described by Lakowicz and co-workers (6),the frequency limit could easily be extended up to 2 GHz. The important point to be gained from Figure 1 is that higher modulation frequencies 0003-2700/88/0360-1622$01.50/0
provide enhanced resolution of Raman scatter from fluorescence. Moreover, if high enough frequencies could be achieved, essentially every fluorescent species would have a 90° phase shift, but Raman would still be phase-shifted Oo. The result would be that several fluorophores in a mixture can be "nulled" simultaneouslyand the Raman spectrum recovered easily. In this correspondence, we demonstrate that good Raman spectra can be acquired from samples containing more than one fluorescing interferant.
EXPERIMENTAL SECTION Rhodamine B, rhodamine 6G (99+%), anthracene,9-phenylanthracene, and 9,10-diphenylanthracene,were from Aldrich and each was used as received. All measurements were performed with a modified SLM 48000 (SLM-Aminco) multifrequency phase-modulation fluorometer. Excitation radiation is provided by a Coherent Model 90-6 argon-ion laser (351.1 and 514.5 nm). Cooling for the argon-ion laser is by an external high-capacity cooling unit (Neslab; Model HX-750). A spectral band-pass of 2 nm was used for the emission monochromator. Modifications to the SLM 48ooo include the addition of thermoeledridy cooled photomultipliertube housings (Thorn EMI), computer control of the frequency synthesizers,and 5OO-MHz frequency synthesizers (PTS, Inc.; Model PTS 500).
RESULTS AND DISCUSSION Figure 2 (curve A) shows a steady-state fluorescence spectrum for 10 nM rhodamine B in water excited at 514.5 nm. One can see visually the characteristic red-orange fluorescence of rhodamine B from this sample. The small shoulder near 625 nm is in small part (6%) from the water Raman. Rhodamine B is an interesting example because its fluorescence lifetime is quite short (1.4ns), significantly less than that of rhodamine 6G (3.7 ns). In spite of this significantly shorter decay time, PRRS is still capable of suppressing even the intense fluorescence from rhodamine B (Figure 2, curve B). Total data collection time for this spectrum was 10 min (200data points). In order to achieve these results, a modulation frequency of 150 MHz was employed. In our case, we have found that there is no real advantage gained by operating above 150 MHz. In fact, poorer results are often obtained because the signal-to-noise ratio becomes poorer. This is entirely due to poorer depth of modulation from the Pockel cell light modulator at higher frequencies. If necessary one could completely eliminate this problem by employing a mode-locked synchronously pumped dye laser system (6). The previous example involved a short-lived single-component interferant; however, one may not always be sure that the fluorescence interference is due simply to a single component. For that reason we studied several mixtures of rhodamine 6G and rhodamine B in water. Typical results are shown in Figure 3. In this case, an aqueous sample containing 5 nM each of rhodamine B and rhodamine 6G was studied. Curve A is the steady-state emission spectrum for the sample and curve B is the phase-resolved Raman spectrum acquired at a modulation frequency of 150 MHz. From this example, PRRS is clearly capable of suppressingthese two components, yielding the unperturbed characteristic Raman spectrum for water. 0 1988 American Chemical Society
Anal. Chem. 1988, 60, 1623-1624
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Figure 1. Theoretical effect of modulation frequency on the recovered Raman signal (enhancement factor) In a phase-resolved Raman experiment. Fluorescence lifetimes of 100 ps, 1 ns, 5 ns, and 10 ns are shown. Note all curves are normalizedto the 10-ns value at 275 MHz.
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Flgure 4. (Curve A) Steady-state emisslon spectrum for an ethanol solution containing 10 nM each of anthracene, 9-phenylanthracene, and 9, lodiphenylanthracene. (Curve B) Phase-resolved Raman spectrum for the same sample at 150 MHz. Scan time is 15 min (100 points).
containing anthracene, 9-phenylanthracene, and 9,lO-diphenylanthracene (each at the 10 nM level). In this case, laser excitation was at 351.1 nm. Again, as in the binary studies, well-resolved Rainan spectra were easily recovered by using the 150-MHz modulation frequency and PRRS. In conclusion, by use of high-frequency modulation, PRRS is capable of resolving the weak Raman scatter from intense fluorescencefrom samples containing mixtures of fluorophores. We are presently attempting to extent high-frequency phase-resolved Raman measurements to solid samples.
Wavelength (nm)
Figure 2. (Curve A) Steady-state emission spectrum for 10 nM aqueous rhodamine B. (Curve B) Phase-reWd Raman spectrum for the same sample at 150 MHz. Scan time is 10 min (200 points).
Registry No. Rhodamine B, 81-88-9;rhodamine 6b, 989-38-8; anthracene, 120-12-7; 9-phenylanthracene, 602-55-1; 9,lO-diphenylanthracene, 1499-10-1.
LITERATURE CITED (1) Olsen, E. D. Modern Optlcal Methods of Analysis; McGraw-Hill: New York, 1975; Chapter 6.
(2) Bright, F. V.; Hieftje, G. M. Appi. Spectrosc. 1986, 4 0 , 583. (3) Spencer, R. D.; Weber, G. Ann. Natl. Acad. Sci. 1969, 158, 361. (4) Demas, J. N.; Keller, R. A. Anal. Chem. 1985, 5 7 , 538. (5) McGown, L. B.; Bright, F. V. CRC Crlt. Rev. Anal. Chem. 1987, 18, 245. (8) Lakowicz, J. R.; Laczko, G.; Gryczynski, I. 6/0Ch8n7/Stty1987, 26. 82.
Frank V. Bright Department of Chemistry State University of New York a t Buffalo Buffalo, New York 14214 525
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Figure 3. (Curve A) Steady-state emission spectrum for an aqueous sample Containing 5 nM each of rhodamine 6G and rhodamine B. (Curve B) Phase-resolved Raman spectrum for the same sample at 150 MHz. Scan time is 25 mln (200 points).
To further demonstrate the multicomponent suppressing abilities of PRRS, we studied ternary mixtures of anthracene derivatives. Figure 4 shows the steady-state (curve A) and phase-resolved (curve B) emission spectra for ethanol solutions
RECEIVED for review November 30, 1987. Accepted March 19,1988. This work was supported by BRSG SO7 RR 07066 awarded by the Biomedical Research Support Grant Program, Division of Resources, National Institutes of Health, by the donors of the Petroleum Research Fund, administered by the American Chemical Society, and by a New Faculty Development Award from the New York State/United University Professions.
Energy Distribution in the Beam of an Infrared Microscope Sir: It is widely recognized that most of the energy in the beam of an infrared (IR) spectrometer is highly concentrated in a small area that is centrally located when the instrument
is in good alignment. In this discussion “homogeneous”refers to the homogeneity or lack thereof of the sample area. When a spectrum of a homogeneous sample is prepared, this dis-
0003-2700/88/0360-1623$01,50/0 0 1988 American Chemical Society
