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Detection

Instrument Effects

Fluorescence signals are influenced by both sample- and instrument-specific contributions. In practice, it is quite difficult to reproduce luminescence data on different instruments or after longer operation time.

A major source of difficulties are instrument-specific contributions to the fluorescence signal. These wavelength-, polarization- and time-dependent quantities reflect

  • the spectral radiance of the excitation light source
  • the transmittance of optical components like lenses, mirrors, filters, monochromator gratings or polarizers in the excitation and emission channel
  • the spectral responsivity of the detection system

The influence of spectral responsivity s(λem) on fluorescence data and the according need for its consideration, i.e., spectral (emission) correction are illustrated in Figure 1 exemplary for a typical organic fluorophore and two spectrofluorometers.

Accordingly, the comparability of fluorescence data and the use of fluorescence spectra for the identification of analytes rely on the knowledge and consideration of s(λem) and its time-dependent changes due to aging of instrument components. The latter makes regular control of s(λem) mandatory but simultaneously comprises an elegant tool for IPV and instrument longterm stability.



Fluorescence Wave

Figure 1. Normalized uncorrected (open symbols) and corrected (full symbols) emission spectra of a typical organic chromophore measured with two different fluorometers. The (at the maxima) normalized spectral responsivities of both instruments are depicted in the inset.


Certified Fluorescence Standard
Determination of the Spectral Responsivity
Kit Spectral Fluorescence Standards: Principle and Properties
Tracability, Certification, and Proviniency Testing
References

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