## Laboratory methods

Commercially available equipment (spectrophotometers) used for the measurement of light absorption, do so in terms of an optical parameter known as the absorbance or optical density: although there is no standard symbol we shall indicate it by D. Although absorbance is one of the most frequently measured parameters in biochemistry and chemistry it does not, unfortunately, appear to have a rigorous definition. It is commonly defined as the logarithm to the base 10 of the ratio of the light intensity, I0, incident on a physical system to the light intensity, I, transmitted by the system

The meaning of the word 'intensity' is not usually given: we shall equate it to radiant flux, F. The incident beam is generally assumed to be parallel. The exact meaning of 'transmitted' is also not stated. If we make the simplest assumption that any light that re-emerges from the system, in any direction, is 'transmitted', then I includes all scattered light as well as that neither absorbed nor scattered. Thus, I is equal to the incident flux minus the absorbed (F0 - Fa), from which, given that Fa/F0 is absorp-tance, A, it follows that

If we can arrange that all the scattered light is included in the measured value of I, then scattering may be ignored (except in so far as it increases the pathlength of the light within the system), and so eqn 1.36 may be considered to apply. Therefore,

i.e. the absorbance is equal to the product of the absorption coefficient of the medium and the pathlength (r) through the system, multiplied by the factor for converting base e to base 10 logarithms. The absorption coefficient may be obtained from the absorbance value using a = 2.303 D/r (3.4)

In a typical spectrophotometer, monochromatic light beams are passed through two transparent glass or quartz cells of known pathlengths, one (the sample cell) containing a solution or suspension of the pigmented material, the other (the blank cell) containing pure solvent or suspending medium. The intensity of the light beam after passing through each cell is measured with an appropriate photoelectric device such as a photomulti-plier. The intensities of the beams that have passed through the blank cell

Blank

cell