Spectrofluorometer limitations

In this detector, the solute is excited with UV radiation and emits radiation at a longer wavelength. The limits of this detection by a spectrofluorometer depend upon the physico-chemical properties of the compound (degree of aromacity), the solvent, and the pH. The eluents (mobile phase) should neither fluoresce nor should they absorb at the excitation and emission wavelengths used. The pH is important because some compounds show fluorescence only in particular ionic forms. There are only a few... 

Carl Zeiss, Inc. also describes a spectrofluorometer system for process monitoring," but it does not currently appear as a standard marketed product on their web site. HORIBA Jobin Yvon also markets a fluorescent process analyzer, but it is a laser-induced time-domain based measurement system tailored for uranium or equivalent analysis." Finally, while numerous miniature spectrofluorometers are also available (Carl Zeiss, StellarNet Inc., Ocean Optics and Avantes), they are not packaged and configured for process applications. Although there is an established need and continued growing interest in realtime process spectrofluorometry, relative to conventional process spectroscopic instruments such as NIR, UV-vis and Raman, commercial process spectrofluorometers are currently available on a very limited basis. 

By reaction of natamycin (or its decomposition products containing the mycosamine group) with NBD-chloride (4-chloro-7-nitrobenzo-2-oxa-l,3-diazole) a fluorescent derivative was obtained. Upon chromatography on silica gel the spots were quantified by means of a spectrofluorometer. The detection limit on the plate was 8 ng [27]. This procedure has been applied for the determination of residual natamycin in food. 

A xenon-arc lamp is used as the source in most spectrofluorometers, since it emits continuously over the range 200-700 nm (see Fig. 9.4C) and hence can be used to obtain fluorescence excitation spectra as well as emission spectra. (Emission spectra for many substances could be obtained with a mercury-arc source, but excitation spectra could not, because the emission is discontinuous and the frequency range is so limited.) However, in uncorrected instruments using a xenon-arc lamp, no correction is made for the variation in intensity of the source with changing wavelength. 

Obviously, a medium-priced spectrofluorometer could have been equipped with a xenon-arc source and used for the analysis, but the filter fluorometer is usually preferred for routine work. In addition, the latter is more sensitive for example, the detection limit for ASA on the Turner spectrofluorometer is 10 M, whereas the detection limit using the Turner filter fluorometer is lO" M. 

In routine analysis for salicylic acid, a medium-priced spectrofluorometer again could have been used. Since the xenon arc has a higher intensity at 308 nm than at 254, a spectrofluorometer with a xenon arc could give a lower detection limit for salicylic acid than would a filter fluorometer. In the analysis for salicylic acid in aspirin tablets, however, it did not prove necessary. The filter fluorometer thus provided a cheaper, more convenient, approach to routine analysis. 

The environmental samples were analyzed for tracer with a Turner 430 Spectrofluorometer at fixed wavelengths (excitation 356 nm emission = 420 nm). Standards were interspersed with samples, and the amount of tracer present calculated by means of a standard curve. The limit of detection of the instrument for this compound was < 5 ppb, allowing detection of levels as low as 150 ng per sample. 

We now describe the individual components of a spec-trofluorometer. The general characteristics of these components are considered, along with the reason for choosing specific components. Understanding the characteristics of these components allows one to understand the cq>abilities and limitations of spectrofluorometers. We will first consider light sources. 

Fluorometers are more sensitive because filters allow more excitation radiation to reach the sample and more emitted radiation to reach the transducer. Thus, a fluorometer can provide lower limits of detection than a spectrofluorometer. In addition, fluorometers are... 

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