Many companies claim high sensitivity for their spectrofluorometers. In their specifications, instrument vendors often cite excitation of de-ionized water at 350 nm, which results in Raman emission at 397 nm (an energy loss of 3380 cm1). This Raman-emission band of water is used, because the signal is red-shifted and weak, thus mimicking weak fluorescence. De-ionized water is easy to prepare, making sample-to-sample reproducibility straightforward.
Other wavelengths, however, also should be considered, because a spectrofluorometer is used across a wide range of wavelengths. Optical design of less-sensitive instruments usually involves lenses and concave gratings, which lose focus at wavelengths other than the design wavelength, degrading instrument performance away from the optimum wavelength.
SPEX spectrofluorometers, however, are free from these artifacts, because their design uses only mirrors and plane gratings. Mirrors and plane gratings remain in focus at all wavelengths, maintaining high performance from the deep-UV to the near-IR. The Raman scatter of water becomes less intense as the excitation wavelength increases. Measurement of a series of Raman spectra across the instruments usable wavelength range is much more relevant as a figure of merit of instrument sensitivity, as the following experiment demonstrates.
Measurements were performed on a FLUOROLOG-3 spectrofluorometer with doublegrating monochromators, a 450-W xenon lamp, and a cooled R928 photomultiplier operated at 950 V in the photon-counting mode. The bandpass was set to 5.0 nm on both excitation and emission spectrometers. The same grating (330-nm blaze, optimized for 500 nm) was used for all scans. Only the integration time varied (see Table 1). The sample was deionized water puri fied in a Millipore Simplicity 185. Excitation of the sample occurred from 250 600 nm, in 50-nm increments. Each emission scan was recorded with a step size of 1 nm. The scans were taken under ambient room conditions.
Results and Discussion
The largest water Raman emissions are the 335-nm, 397-nm, and 463-nm peaks (see Figure 1), which also have the best signal-to-noise ratio, S/N (see Table 2). As the incident wavelength increases, the Raman peaks are displaced farther from the incident wavelength, but the energy loss is a constant 3380 cm1. Raman peaks are also visible at 275 nm, 531 nm, and 693 nm. The emission spectra are re-scaled to reveal the Raman bands more clearly at 667 nm and 753 nm (see Figure 2). These results demonstrate the high sensitivity of the SPEXFLUOROLOG-3 even at these long wavelengths.
Water-Raman transitions are detectable across a large spectral range. Therefore, the SPEXFLUOROLOG-3 demonstrates high sensitivity to fluorescence signals throughout the UV-visible region; see if your system can handle this task. Kinematically mounted gratings allow optimization of the spectrofluorometer for custom applications at various wavelengths. Gratings, blazed at different wavelengths and optimized for different spectral regions, are changed easily with instant alignment. The SPEX FLUOROLOG-3 reaches the statistical noise limit: this means that SPEX gives you the best system possible.