UV-VIS Spectroscopy

Spectroscopy is measurement and interpretation of electromagnetic radiation absorbed, scattered or emitted by atoms, molecules or other chemicals species. Absorption and emission is associated with changes in energy states of the interacting chemical species and since each species has characteristic energy states, spectroscopy can be used to identify the interacting specimen. [1]

UV and Vis spectra of organic compounds are associated with transition between electronic energy levels. The transition are generally between a bonding or lone-pair orbital and an unfilled non bonding or antibonding orbital. The fundamental law underlying the practice of absorption photometry called Beer’s law, “The absorption is proportional to the number of absorbing molecules”. When a beam monochromatic light, previously rended plane-parallel, enters an absorbing medium at right angles of plane, parallel surface of the medium, the rate of decrease in radiant power with the length of light path (cuvette interior) b, or with the concentration of absorbing material C (in grams per liter) will follow the exponential progression:
           
T = 10^-A

Certain thickness of material absorb half the radiant energy, then an equal thickness of material which follow will absorb half of the remainder, and so on. Doubling the concentration of an absorbing material has the same effect as doubling the path length. The absorbent is given by:

A=abC

Where a is the absorptivity of component interest in solution. The absorptivity constant dependent upon the wavelength of the radiation and the nature of the material. The product of absorptivity and molecular weight is called molar absorptivity and is given the symbol ε. The sensitivity of the colorimetry method is governed basically by the molar absorptivity of the color species formed, but can be increased by developed the small a volume as possible.[1]

The absorption of UV or visible radiation corresponds to the excitation of outer electrons. There are three types of electronic transition which can be considered [2]
1. Transitions involving p, s, and n electrons
2. Transitions involving charge-transfer electrons
3. Transitions involving d and f electrons (not covered in this Unit)
Absorbing species containing p, s, and n electrons Absorption of ultraviolet and visible radiation in organic molecules is restricted to certain functional groups (chromophores) that contain valence electrons of low excitation energy. The spectrum of a molecule containing these chromophores is complex. This is because the superposition of rotational and vibrational transitions on the electronic transitions gives a combination of overlapping lines. This appears as a continuous absorption band. [3]

Instrumentation of UV-Vis spectrophotometer consists of: [3]
1.  The light source. The tungsten filament lamp and hydrogen discharge lamp are fitted.
2.  Monocromator. The beam is dispersed into a spectrum by 30-degree prism of crystal quartz or fused silica, and is reflected back by an aluminized coating back of the prism. The monocromator involves 3 irrelated factors; resolution, light gathering power, and purity of light output.
3.The absorbing specimen. Monochromatic radiation emerging from the slit 2 passes trough the cell chamber which will house four cell of the length up to 4 cm. They are made of fused silica, which is transparent to about 1800Å, and are mounted on so that anyone maybe place on the beam.
4.Photocell. Photo emissive they are mounted behind the specimen cells. The range is covered by two cells with different cathode material. The beam is directed to one or the other by rotable mirror.
5.Recording system. In recording there is always some time lag between recorder reading and actual value. Scanning speed should be selected to ensure that the detecting system can follow the signal from narrow emission lines of absorption bands.

Reference:
1. Willard, Merrit, and Dean (1974), Instrumental Methods of Analysis 5th Edition, New York.
2. UV-Vis. Absorption Spectroscopy - Theoretical Principles, Chemistry Division 3. Homepage, Sheffield Hallam University, School of Science and Mathematics website
3. Browning, D.R, Spectroscopy, McGraw Hill, London.