Spectrophotometer is an instrument consisting of spectro photometer and photometer. Spectrophotometer produces rays from spectrum with wavelength and photometer gauges for measure intensity of light which is transmitted or absorbed. So, the spectrophotometer is used to measure relatively energy if the energy is transmitted.
The spectrophotometer method is based on conversion of light polikromatis from the light source to be monochromatic light by a monochromator, and then it is passed through filter and will pass through the sample where some of light will be absorbed and some will be transmitted. This light will be detected by detector and it is amplified by the amplifier. The result will be recorded by recorder.
The relationship between radiation absorption with wavelength through the absorbing medium was first expressed by Bouguer, although sometimes it is ascribed to Lambert that his statement reads: “The power of radiation that is transmitted decreases exponentially if thick absorbing medium increases arithmetically. While Beer issued contradictory statements on Lambert’s statement that the light which is used to be monochromatic, otherwise you will get two values of absorbance at two wavelengths.”
If a system follows Beer’s statement, a graph of absorbance against concentration will produce straight line, where the absorbance is directly proportional to concentration. Lambert and Beer’s law indicates the relationship as follows:
a = constant absorption
b = distance optical
c = concentration
T = transmission
A = absorbance