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Transmissionof light

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Light is a form of electromagnetic radiation, usually a mixture of waves having different wavelengths. The wavelength of light, expressed by the symbol λ, is defined as the distance between two crests (or troughs) of a wave, measured in the direction of its progression. The unit used is the nanometre (nm, 10-9 m). Light that the human eye can sense is called visible light. Each colour that we perceive corresponds to a certain wavelength band in the 400-700 nm region. Spectrophotometry in its biochemical applications is generally concerned with the ultraviolet (UV, 185-400 nm), visible (400-700 nm) and infrared (700-15 000 nm) regions of the electromagnetic radiation spectrum, the former two being most common in laboratory practice. The wavelength of light is inversely related to its energy (E), according to the equation: . . . E = ch/ λ . . . where c denotes the speed of light, and h is Planck’s constant. UV radiation, therefore, has greater energy than the visible, and visible radiation has greater energy than the infrared. Light of certain wavelengths can be selectively absorbed by a substance according to its molecular structure. Absorption of light energy occurs when the incident photon carries energy equal to the difference in energy between two allowed states of the valency electrons, the photon promoting the transition of an electron from the lower to the higher energy state. Thus biochemical spectrophotometry may be referred to as electronic absorption spectroscopy. The excited electrons afterwards lose energy by the process of heat radiation, and return to the initial ground state. An absorption spectrum is obtained by successively changing the wavelength of monochromatic light falling on the substance, and recording the change of light absorption. Spectra are presented by plotting the wavelengths (generally nm or μm) on the abscissa and the degree of absorption (transmittance or absorbance) on the ordinate. For more information on the theory of light absorption, see Brown (1) and Chapters 2, 3 and 4. The most widespread use of UV and visible spectroscopy in biochemistry is in the quantitative determination of absorbing species (chromophores), known as spectrophotometry.

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