10.3.3: The Detection Advantage
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Fluorescence methods are among the most sensitive analytical methods available to chemists. In fact with a specialized instrument setup, one can detect the fluorescence from a single molecule. The harder trick in this experiment is isolating the single fluorophore in the focal area of the instrument.
In general and for experiments routinely available instrumentation, fluorescence methods have sensitivities that are one to three orders of magnitude better that absorbance measurements. The enhanced sensitivity for fluorescence arises from the difference between the wavelength of emission relative to wavelength of excitation and the fact that the concentration related parameter for fluorescence, the fluorescence intensity F, can be measured independent of the power of the optical source, P0. In contrast, an absorbance measurement requires evaluation of both P0 and P, because absorbance, which according to Beer's Law is proportional to concentration, is dependent on the ratio of the two quantities.
Consider Figure \(\PageIndex{1}\) illustrating the difference between the absorbance and fluorescence experiments.
As we saw in Chapter 8, as the concentration of the absorbing species decreases the difference between P0 and P gets smaller and the uncertainty with measuring this difference increases. This lead to a lower limit of detection for a strongly absorbing species with a molar absorptivity of 50,000 liter/(mole cm) to approximately 10-6 M.
In the case of fluorescence there is no light to detect at the emission wavelength (not even scattered light at that wavelength) unless the fluorophore is present in the sample. One can either collect the fluorescence signal, F, for a longer time or increase P0 to detect measurable signal. Consequently the lower limit of detection in fluorescence is on the of 10-10 M.
It is also true that the minimum volume of required sample is much less for fluorescence than for absorbance. However, the upper limit in terms of concentration for absorbance is much larger and the accuracy and precision for quantitative fluorescence methods is poorer by a factor of about 5X.