Biochemical Techniques and Instrumentation

1.2.2. Double beam UV-Vis Spectrophotometer

In double beam spectrophotometer, the incoming light beam is split into two parts by a half mirror. One beam passes through the sample, the other through a control. This eliminates the problems of variations in light intensity. The measure absorbance is the difference between the two transmitted beams of light recorded. One detector measures the incoming and transmitted intensity alternately. Sometimes, a second detector is separately used to measure the intensity of the incoming beam. The instrument changes the light source from visible to UV light at about 350 nm by a mechanically moving mirror.

The main parts of the UV-Vis double beam spectrophotometers are light source, monochromator, sample cell, detector and integrator.

Schematic diagram of UV-Vis spectrophotometer