Basis of Derivative Spectrophotometry
Photobiological processes occur under the influence of light of ultraviolet (UV), visible, and near infrared spectral regions. Generally, values of light flux intensity, I, and wavelength, λ are used in optical measurements. The frequency index \( \bar{\n
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Basis of Derivative Spectrophotometry
2.1
The Main Law of Light Absorption by a Substance
Photobiological processes occur under the influence of light of ultraviolet (UV), visible, and near infrared spectral regions. Generally, values of light flux intensity, I, and wavelength, l are used in optical measurements. The frequency index n is also considered to characterize an absorbed light. Frequency is expressed in reciprocal seconds [c1] and presents itself as the ratio of a radiation velocity c to a wavelength l [cm or nm]: n ¼ c=l ¼ 31017 =lnm ; where c ¼ 3 1010 cm c1 or 3 1017 nm c1, the velocity of light. The frequently used index is the wavenumber n equal to the number of waves in 1 cm. The wavenumber is a reciprocal wavelength expressed in reciprocal centimeters: ½cm1 : n ¼ 1=lcm ¼ 107 =lnm Thus, n ¼ 40; 000 cm1, when l 250 nm. A number of spectrophotometers have wavenumbers on the scale dials. For example, the wavelength of the cadmium red ˚ (angstroms). line, as accepted by international agreement, is equal to 6438.4696 A ˚ So 1 A is 1/6438.4696 part of cadmium red line. Only absorbed quanta can realize photochemical impact and the reader should remember that a spectral region of a photobiological process is conditioned by the absorption spectrum of a substance involved in this process. The extinction law for actinic monochromatic light absorbed by a substance layer can be written in the exponential or logarithmic form: I ¼ I0 10eCd ;
V.S. Saakov et al., Derivative Spectrophotometry and Electron Spin Resonance (ESR) Spectroscopy for Ecological and Biological Questions, DOI 10.1007/978-3-7091-1007-2_2, # Springer-Verlag Wien 2013
(2.1)
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lgðI0 =IÞ ¼ eCd;
Basis of Derivative Spectrophotometry
(2.2)
where I0 and I are light intensity before and after passing through a layer of substance, of solution, or a leaf, e is a coefficient depending on wavelength and nature of a substance, C is a concentration of a light-absorbing substance in medium, through which light passes, and d the layer thickness (length of the light path in a cuvette, an optical cell). If the concentration of solution is expressed in moles, then e is called the molar extinction coefficient (MEC). The value of e is numerically equal to the optical density of solution of concentration 1 mole/L at cuvette thickness 1 cm [L mole1 cm1]. Often solution concentration is expressed as a percentage and, instead of e, the so-called specific extinction is used (E1% 1cm), equal numerically to an optical density of 1% substance solution at the cuvette thickness 1 cm. This value is necessary to determine and to use during instrument calibration for calculation of absolute concentration of a substance in solution. The ratio of passing and incident light intensities is called the transmission and calculated as T ¼ I=I0 : The same ratio expressed as a percentage is placed at the left scale of a recording card in Russian spectrophotometers of types SP-14 and SP-18 and some European ones. It is called “the transmission
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