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What is UV - Vis Radiation? Ultraviolet radiation is part of the electromagnetic spectrum, just like visible light, infra red, microwave and radio. In UV-Vis spectroscopy we are using the radiation of our visible range (700-400nm) and the ultraviolet range (400-100nm) to probe the analyte for structural clues.
Fig.1: the electromagnetic spectrum As you can see, this range of radiation has more energy than IR and radio frequencies (RF, used for NMR). the actual energy of a photon of radiation of a certain frequency can be calculated thus:
E= hv Fig.2: energy of radiation, where e= energy (J), h= Planck's constant (6.626x10-34Js), v = frequency (Hz) For the UV-Vis range, energies run from 171kJ to 1,197kJ per mol of photons (for 700nm and 100nm respectively). This energy is sufficient to excite certain electrons and promote them to a higher orbital. As orbitals are quantised, only certain transitions are possible in this energy range, as shown below Fig.3: orbital promotion Above, if the transition of E1 to E2 is within the range of 171kJmol-1 to 1.19MJmol-1, the electron will be promoted. If say, the transition E3 to E4 requires more energy (i.e. the orbitals are not close in energy), perhaps 2MJmol-1, the transition will not occur. It happens that π orbital transitions (from π to π*) are in the UV energy range, so aromatic and unsaturated systems give strong responses (they are strong chromophores). Weaker absorptions include double bonds (at around 190nm), such as ketones, esters, amides, and nitro-compounds. Weaker still are the responses seen by heteroatoms such as nitrogen and oxygen (for instance in amines and alcohols respectively, due to their lone pairs on the hetero atom, seen weakly at 185-195nm). Carbon-carbon and carbon-hydrogen bond electrons do not absorb at all well in the UV range, so alkanes are not usually seen (these are in the range of 150nm) but are very very weak. Note that as solvents are in such a large excess, that UV-vis instruments often cannot remove the reference sample totally. Therefore any peaks below about 220nm should be regarded with care. If possible, avoid using anything in this range as a calibration wavelength. Since the transitions require energy which the molecule absorbs from the incident beam of UV radiation, the light that passes through the sample will have less (or none) of light of that particular energy, thus it is possible to calculate the differences in the incident and transmitted beam, to see which frequencies have been absorbed by the molecule. As aforementioned, these transitions are quantised so information about the analyte can be derived by the frequencies of the light absorbed (for example, if the sample has an absorbance at 254nm, it is thought to have a delocalised pi system, such as a phenyl ring or other aromatic system). UV-vis, as with other spectrophotometric techniques can be used in a quantitative way (see quantitative UV section), due to absorbance's dependence on concentration, as denoted by Beer-Lambert's Law.
A = εcl Fig.4: Beer-Lambert's law Above it can be seen that absorbance (A) is the product of molar extinction coefficient (ε, also called the molar absorptivity) concentration (c) and path length (l, this is the length of the sample the light has to permeate. Usually it is a 1cm cuvette).The molar extinction coefficient is unique to each compound and does not change with concentration. The Beer-Lambert Law only applies to low concentrations. At higher concentrations, shadowing effects stop molecules being able to absorb the radiation (molecules at the far side of the cuvette are unable to absorbe any radiation, as they are in the shadow of molecules at the near side of the cuvette, and hence the results obtained do not represent the whole sample).
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