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Study Notes: The Spectrophotometer

The spectrophotometer is an analytical instrument that measures the changes in a beam of light of a given wavelength that passes through a solution sample held in a special receptacle called a cuvette.

Image of a cuvette. The cuvette is usually 1 cm square with two frosted sides and two clear window sides. The spectrophotometer is calibrated to a light path of 1 cm across the cuvette. The window sides allow the transmission of the beam of light and are calibrated so that the cuvette will only work if placed into the cuvette holder in the correct orientation. A mark on the outside of the cuvette is aligned with a mark on the spectrophotometer to allow correct orientation of the cuvette in the instrument.

The spectrophotometer is able to use a beam of light in the UV-Visible spectrum that is selected by the operator to suit the requirements of testing. The wavelength of light is measured in nanometres (10-9 metre).

The spectrophotometer either measures the amount of light transmitted through a solution (called transmittance) or the amount of light absorbed by a solution (called absorbance or extinction).

At SimuLab we use transmittance. The spectrophotometer is first zeroed (with no cuvette or sample in the sample holder) then a blank solution (ie the substrate containing the test sample) is used to calibrate the instrument to 100% transmittance. That is, no light of that wavelength is absorbed by the blank solution.

The calibration of the instrument is then checked using Calibration Check Standards to ensure that the instrument has been calibrated correctly and is ready to use to measure unknown samples.

When measuring unknown samples, a reduction in transmission is then directly related to the increase in concentration of the sample of interest.

Image of a labelled spectrophotometer.

The important parts of the spectrophotometer (for the operator) are:

On/Off button turns the machine on and off.
Pilot light indicates that the machine is turned on.
Wavelength display indicates the selected wavelength in nm (nanometre).
Digital readout indicates the % Transmission of the light beam.
Cuvette sample holder holds the cuvette in a light proof container when the lid is closed. It is important that the lid is closed, as extraneous light from the environment will cause the instrument to misread.

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