Chimactiv - Interactive numerical educational resources for the analysis of complex media

A spectrophotometer

A "single-beam" spectrophotometer is made up of:

a polychromatic source
a wavelength selector and eventually a source selector
a sample compartment
a detector
a screen and/or a computer.

Schematic diagram of a spectrophotometer. On the left, a light source emits a polychromatic light through the monochromator. The latter decomposes the different wavelengths and allows to select one of them. The selected beam passes through the sample tank before reaching the detector. The signal of the latter is amplified before being displayed.

For liquids, cuvettes with a defined path length can be used. They can be in quartz, glass or plastic, for example, depending on the sample type.


1 mm wide glass	10 mm wide glass	10 mm wide "quartz"	50 mm wide glass	10 mm wide glass, small volume	10 mm wide glass, very small volume	10 mm wide plastic, shrinked

Powder samples are placed in a special sample holder (integrating sphere).

Schematic representation of a spectrophotometer

Click on each element to see the details

Double-beam spectrophotometers are more sophisticated and able to simultaneously measure a sample and a reference. I₀ and I are both determined and compared to obtain a resulting spectrum.

How does a spectrophotometer work?

"White" light is obtained from a polychromatic source, then a monochromator extracts the radiation at the wavelength requested by the user. Beam intensity is measured after it has passed through the sample in the cuvette. This intensity is compared to a reference value – the resulting value displayed by the instrument reflects this comparison. Thus, for a single-beam spectrophotometer, the reference spectrum is recorded with the solution (solvent or buffer) but without the sample. For a double-beam spectrophotometer, one cuvette contains the sample and the other one only contains the solution: the difference between both absorbances is then directly done by the system.