Absorption of UV-visible light

La lumiere (...) donne la couleur et 1 eclat a toutes les productions de la nature et de l art elle multiplie l univers en le peignant dans les yeux de tout ce qui respire. 

The aim of this chapter is to recall the basic principles of light absorption by molecules. The reader is referred to more specialized books for further details. 

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Principle An UV-detector is based on the principle of absorption of UV visible light from the effluent emerging out of the column and passed though a photocell placed in the radiation beam. 

Figure 1. Electronic excitation from the highest occupied molecular orbital to the lowest unoccupied molecular orbital. The absorption of UV-visible light promotes a molecule from its ground electronic state to an excited state. The excited state shown is a singlet excited state where all electron spins are paired.

Derivatizations for HPLC are de.signed mainly to improve the limit of detection, permitting the use of highly sensitive or selective detectors inapplicable to the analytes themselves. Enhanced absorption of UV/visible light is achieved by the introduction of chroniophoric groups. Analytes can also be rendered fluorescent by the introduction of fluorophoric groups. 

Just as the absorption of UV or visible light causes electrons to excite between different electronic quantum states, so absorption of infrared photons causes excitation between allowed vibrational states, and absorbing microwave radiation causes excitation between allowed rotational states in the absorbing molecule. As a crude physical representation, these quantum states correspond to different angular velocities of rotation, so absorption of two photons of microwave radiation by a molecule results in a rotation that is twice as rapid as following absorption of one photon. 

The depth-profile of photon absorption is analogous to that for UV-visible light, i.e. I = Io exp(-Ad), where the mass energy absorption coefficient, u/g is used instead of the extinction coefficient. Particulate energy absorption can be described by relative stopping powers. 

Fluorescence spectroscopy is commonly used to characterize fluorescence effects in the UV and visual range of the electromagnetic spectrum. Such fluorescence is caused by the fact that the absorption of UV or visible light of specific wavelengths causes excitation of electrons within a molecule. If radiating relaxation occurs directly from the singlet Sj state, the process is called fluorescence. 

Detect the two forms, A (colourless) and B (coloured), using light of a lower energy than the UV-Visible light used for the interconversion (A into B, B into A). The readout can be measured as the difference in the reflecting light intensity of the laser e.g. diode), or by fluorescence or infrared absorption signals in the two... 

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