Question

Which method would be better for determining very low concentrations of a particular analyte in solution, absorbance or fluorescence spectrophotometry? Why?

Which is more selective, absorbance or fluorescence spectrophotometry and why?

What is the difference between absorbance, fluorescence and phosphorescence?

Answer 1

Fluorescence spectrophotometry is better than absorbance spectrophotometry for determining very low concentrations in solution, given the analyte is fluorescent.

Absorbance is measured as the difference in intensity between light passing through the reference and the sample. In fluorescence the intensity is measured directly, without comparison with a reference beam. Fluorescence has greater limits of detection, while potentially using less sample material. This advantage is due to measurement of the fluorescence relative to a dark background, as compared to the bright reference beam in an absorbance measurement. It is relatively easy to detect low levels of light accurately, rather than the very small difference between the absorbances.

Only molecules that fluoresce are detected by fluorescence spectroscopy, resulting in greater selectivity compared with UV/Vis absorption, which does not discriminate between the sample of interest and contaminants that absorb at the same wavelength.

Absorbance is a measure of the capacity of a substance to absorb light of a specified wavelength.

A = -logT , where T is transmittance of the substance/solution.

Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. Fluorescence occurs when an orbital electron of a molecule or atom, relaxes to its ground state by emitting a photon from an excited singlet state.

Whereas, phosphorescence is a process in which energy absorbed by a substance is released relatively slowly in the form of light, unlike fluorescence in which light is emitted as soon as radiation is absorbed (very short time scale).