Scattering (Rayleigh and Raman) and fluorescence are two common light signals that frequently occur together, confusing the researchers and graduate students experimenting in molecular spectroscopy laboratories. This report is a brief study presenting a clear discrimination between the two signals mentioned, employing a common spectrofluorometer such as the PerkinElmer LS 55. Even better, the resonance Raman signal of a molecule (e.g., acetone) can be obtained elegantly using the same instrument.
When photons fall on organic molecules (benzene, acetone, coumarin, etc.), some get scattered, while others are absorbed. Among the scattered signals, some have the same wavelength of the incident photon (termed Rayleigh scattering signals), some have wavelength longer than the incident light (called Raman Stokes signal), and only very few photons have a shorter wavelength (called Raman anti-Stokes signal) [
Some molecules which absorb the photons produce fluorescence; this is a reemission process, a phenomenon from one quantum state to another one. A molecule (i.e., acetone) is at the ground state electronic level S0, which consists of manifold vibro-roto energy sublevels (see Figure
Schematic of energy level diagram of a molecule. S0 and S1 are electronic energy levels of a molecule, with vibration levels
Such absorption and reemission (fluorescence) are reasonably strong only in a few specific set of molecules, like dyes, which have a chain of alternate single and double levels, called conjugation. Most spectrofluorometers, commonly employed in research labs, are used only for the studies mentioned above.
In contrast, scattering is generally nonspecific, implying that light of any wavelength can interact with any molecule and undergo scattering. In this instance, if a photon of frequency
It is very significant that for a given molecule, both types of scattering can be observed by employing UV, VIS, or IR radiation, because the virtual level can be found anywhere. On the other hand, the shift in the scattered radiation
Scattering is always from a real quantum state to a virtual state whereas fluorescence is a phenomenon that occurs between two quantum states; hence, the latter is 10–100 times stronger than the former.
In order to obtain the Raman signal reasonably comparable to the fluorescence signal, one must resort to resonance Raman scattering [
Many new reviews shows ever increasing applications of Raman scattering in fields such as material science from bulk to nano. In nanotechnology, new techniques such as nano-Raman techniques based on aperture scanning near-field optical microscopy (SNOM) are maturing and diversifying [
The PerkinElmer model LS 55 was employed throughout this experiment; however, any other commonly available spectrofluorometer (Horiba, Hitachi, or Shimatsu), with dual gratings, one for excitation wavelength selection and another for emission band scanning, will be equally suitable.
Acetone (analytical grade) quality was taken in a transparent quartz cuvette of 1 cm path length and loaded into the cell compartment of the instrument. Excitation wavelength was arbitrarily selected as 330 nm; width was 10 nm in excitation as well as emission slits. The emission (synonymous with fluorescence in this case) spectrum was scanned from 350 nm to 600 nm and is presented in Figure
The fluorescence spectra of acetone with a peak at 413 nm for different wavelengths of excitation; on its left side is the excitation spectra with a peak at 327 nm.
In this case, the emission grating was fixed at 413 nm, which was the emission peak of acetone and the excitation grating was rotated from 250 nm to 390 nm to obtain the excitation band of acetone, which had a peak at 327 nm with FWHM of 70 nm. This corresponded to the S0-S1 absorption spectrum of acetone, obtainable from any UV-VIS spectrophotometer.
The excitation band indicates that if the excitation had been induced at 327 nm, the emission peak would occur at 413 nm, with the spectral profile of emission band becoming a mirror image of the excitation band, with the dotted line acting as the mirror as shown in Figure
Utilizing the same acetone sample and excitation at 330 nm, emission spectra were obtained for 10, 5, and 3 nm spectral widths, in excitation as well as emission slits. The spectra in Figure
Acetone fluorescence with different slit widths.
To obtain the Rayleigh scattering spectra of acetone, the excitation grating was fixed at 330 nm; the slit width was 10 nm in each slit and the emission grating was scanned from 200 nm to 700 nm. The spectrum, as shown in Figure
Rayleigh scattering and fluorescence signal with different slit widths (for acetone). (a) Black line and (b) blue line.
The first band, without experiencing any change in wavelength, is the Rayleigh scattering (please note, it should have occurred at 330 nm but instead was seen at 338 nm due to instrumental artifacts); the 410 nm band is the fluorescence band of acetone; the band at 673 nm is due to the Rayleigh scattering occurring in the second order of grating (this is an experimental unavailable artifact; if only a prism had been used instead of grating for dispersion, it would have disappeared).
For the same sample and scattering setup arrangement, the experiment was repeated with slit widths = 5 nm in each. As shown in Figure
Scattering is, in fact, an irregular reflection at the molecular surface, very similar to a tennis ball bouncing off a racquet. What comes, goes off in a different direction, with minimal or no interaction. In contrast, during fluorescence, an incident photon is absorbed, and the energy gets thermalized in a few picoseconds and is then given off by the molecule, with its fingerprint, within the time span of a few nanoseconds.
For measuring the Raman spectral features of acetone, the excitation wavelength was fixed at 450 nm and the emission grating was scanned from 485 nm to 600 nm. As evident from Figure
Resonance Raman spectra for acetone.
To confirm this, the experiment was repeated with the excitation at 425 nm and scanning was done from 440 nm to 600 nm. The Raman signal now appeared at 489.5 nm and the Raman shift was
The most significant feature of this part of the experiment is as follows: as the excitation was moved from 450 nm to 315 nm, over a range of 135 nm, the Raman signal intensity changed from 9 to 160 (in arbitrary unit), an increase by 17 times. Apparently, 340 nm represents the near resonance Raman spectra of acetone. When the excitation goes to 325 nm, the Raman signal occurring at 350 nm was comparable in intensity to the fluorescence at 413 nm, representing the resonance Raman excitation; for excitation at 315, the Raman signal occurred at 352 nm, with the intensity of 120. For excitations with a wavelength lower than this, the acetone produced only a fluorescent broad band with a peak of 413 nm, as discussed earlier. Or, in other words, the best region to obtain resonance Raman is the valley between the excitation and emission bands.
Table
Resonance Raman spectra of acetone.
Number | Excitation wavelength |
Excitation wave number |
Raman signal | Raman signal | Raman shift |
Intensity |
---|---|---|---|---|---|---|
In wavelength |
In wave number |
|||||
1 | 450 | 22222 | 519.6 | 19246 | 2976 | 10.7 |
2 | 425 | 23529 | 489.5 | 20429 | 3100 | 17 |
3 | 400 | 25000 | 455 | 21978 | 3022 | 25.5 |
4 | 375 | 26667 | 424 | 23585 | 3082 | 49 |
5 | 340 | 29412 | 380.4 | 26288 | 3124 | 95 |
6 | 337 | 29673 | 382.6 | 26136 | 3537 | 138 |
7 | 325 | 30769 | 370 | 27027 | 3742 | 158 |
8 | 315 | 31746 | 354 | 28248 | 3498 | 135.3 |
|
|
The next experiment was performed with reduced slit widths. Figure
Raman spectra of acetone at different slit widths. (a) Blue line and (b) black line.
In fact, when the Nd:YAG laser (at 532 nm) was used to produce Raman signal from acetone, a sharp signal at 630 nm of FWHM of 1 nm or less was obtained [
Another interesting experiment was performed to highlight the differences in the polarization properties.
Figure
Acetone fluorescence spectra for different positions of polarizer.
Figure
Polarization of Rayleigh scattering of acetone. (a) Black line; (b) blue and red line; (c) green and orange line.
Figure
Raman spectra of benzene at different excitation wavelengths. (a) is black line.
Raman spectra for benzene.
Number | Excitation wavelength |
Excitation wave number |
Raman signal | Raman signal | Raman shift |
Intensity |
---|---|---|---|---|---|---|
In wavelength |
In wave number |
|||||
1 | 475 | 21053 | 501 | 19960 | 1093 | 6 |
556 | 17986 | 3067 | 3.7 | |||
|
||||||
2 | 450 | 22222 | 475.3 | 21039 | 1183 | 9.9 |
525 | 19048 | 3174 | 9.8 | |||
|
||||||
3 | 425 | 23529 | 445.8 | 22472 | 1057 | 14.9 |
490 | 20408 | 3121 | 15.5 |
Figure
Polarized Raman spectra for benzene.
It is noteworthy that all scattering exhibits a high degree of polarization, because it changes both direction and polarization.
Utilizing a spectrofluorometer commonly available in any graduate laboratory, a distinction between most of the spectral features of fluorescence and scattering has been clearly demonstrated (Rayleigh and Raman). It has elegantly been shown that the valley between the two mountains of absorption and emission is the best region to grope for the resonance Raman spectra. It has also been shown that the scattered signals are strongly polarized and that their spectral widths are determined most often by the width of the incident signal. In contrast, fluorescence is neither polarized nor dependent on the incident signal. Further, scattering is many times more common than fluorescence in general; however, for a few select, conjugated molecules like dyes, the fluorescence is tremendously high that it is almost impossible to observe the scattering signals. On the other hand, in the weakly fluorescent molecules like acetone or benzene, the scattering signals are comparable to, and sometimes even higher than, the fluorescence.
The authors declare that they have no conflicts of interest.
The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through the Research Group Project no. “RGP-223.”