When filling a cuvette with distilled water and measuring it with a spectrofluor

Question

When filling a cuvette with distilled water and measuring it with a spectrofluorometer, upon excitation at 350nm, a band around 397nm appears. This is either a fluorescence band or a Raman band. Suggest what you can do experimentally to identify which type of band it is, and why.



"If someone could help explain an appropriate experimental technique to me it would be greatly appreciated. I am currently trying to grasp the concepts of Rayleigh and Raman scattering and determining if they are relevant to this question along with excitation and emission spectra as possible techniques.
It is my understanding that for Rayleigh, excitation comes at 400nm and you are able to determine the time required for scattering and fluorescence is ~10^_8 - 10^-4.
And Raman scattering is used when a weak, but reproducible peak is observed in both solutions under observation.
Also, could Time-resolved Fluorescence Immunoassay be used? Sorry for all of the extra imput, IF anyone can help me piece this together I will rate highly!"

Explanation / Answer

FT-Raman spectra of some calcium phosphates, calcium carbonate and calcium hydroxide are reported. Comparing with dispersive Raman spectra, it is shown that some strong bands in the FT-Raman spectra of these compounds which have been erroneously assigned to Ramanbands, are in fact fluorescence emission bands. The origin of these fluorescencebands have been discussed in terms of rare earth impurities in a particular structure. It is further concluded that in using FT-Raman spectroscopy of minerals, care should be taken in not confusing the fluorescencebands with Raman scattering bands. Among instrumental techniques, fluorescence spectroscopy is recognized as one of the more sensitive. In fluorescence, the intensity of the emission of the sample is measured. The reason for the high sensitivity of fluorescence techniques is that the emission signal is measured above a low background level. This is inherently more sensitive than comparing two relatively large signals as in absorption spectroscopy. The sensitivity of fluorescence techniques is as much as 1000 times more sensitive than absorption spectroscopy. A spectrofluorometer with high sensitivity is an asset for the researcher. It is obvious that an instrument with high sensitivity will be able to perform experiments with the fluorescing species at a low concentration. For analytical measurements, sensitivity determines the detection limit of a material. For research in life science, it may be important to work at concentration levels that are very low. This is almost always the case in biomedical research. A more subtle requirement is the need to measure changes in fluorescence during the course of an experiment. Even though the gross signal levels may be high, the changes in the signal may be quite small. The ability to reliably measure these small changes is directly dependent upon the sensitivity of the fluorometer. The measurement of sensitivity has been discussed in the literature [1]. Unfortunately, there is little consensus on a standard procedure for determining this important capability. This paper discusses the measurement of sensitivity and details two methods that are applicable to any instrument. The Spectrofluorometer The sensitivity of fluorescence is dependent on both the fluorophore and the instrument. The response of a fluorophore will depend on the molar absorptivity and the quantum yield. These factors are, in general, beyond the control of the analyst. The sensitivity of a spectrofluorometer depends on a number of factors. Instrumental contributions to sensitivity are described below. 1. Source intensity. In general, a brighter excitation source will result in brighter emission. The source for most fluorometers is a xenon arc, which has a high intensity between 200-900 nm, the spectral region where most fluorescence experiments are performed. While a high power arc lamp is good for highest intensity, a more important criteria is the brightness of the lamp. The brightness is a function of both the power and the size of the arc. Arc lamps for commercial fluorescence systems range from 75-450 watts. 2. Efficiency of the optical system. The light collection efficiency is a function of two factors. A high efficiency optical design is characterized by a low f number. For example, f/2 is more efficient, or faster, than f/4.5. It is imperative that the various optical components (e.g., source, monochromators, sample cuvette position, and detectors) are optically matched. A high speed design, however, is prone to high stray light. Also, the gratings, mirrors, and lenses that are incorporated into the spectrofluorometer all have associated optical losses and will decrease the intensity of the light passing through the system. The number of optical elements, the thickness of lenses, and the coatings on mirrors and lenses will affect the throughput. 3. Spectral bandpass of the monochromators. The bandpass of commercial fluorometers may be varied between 0.5-30 nm. Doubling the bandpass of a monochromator will increase the throughput of light by a factor of four. Resolution, however, is worse at high bandpass. The bandpass may be adjusted by the analyst to balance sensitivity and resolution. 4. Efficiency of the detector. Both analog and photon counting methods of detection are used in commercial instruments In general, photon counting is considered to be slightly more sensitive. Both methods are subject to degradation because of noise in the electronics. It is the combination of the effects from all of these contributions that determine the sensitivity of the fluorescence spectrometer. It follows, then, that the most appropriate way to quantify the sensitivity of a spectrofluorometer is to measure a standard sample using the complete instrument system.

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