The easiest means of accomplishing this is to use either a dual beam spectrophotometer or a computer controlled instrument. In either event, the baseline must be continously re-read as the wavelength is altered. To use a single beam spectrophotometer, the machine is zeroed first, the wavelength is set, the blank is adjusted and then the sample is inserted and read. This procedure continues until all wavelengths to be scanned have been read. In this procedure, the sample remains the same, but the wavelength is adjusted.
Compounds have differing absorption coefficients for each wavelength.
Thus, each time the wavelength is altered, the instrument must be recalibrated. A dual beam spectrophotometer divides the light into two paths. One beam is used to pass through a blank, while the remaining beam passes through the sample. Thus, the machine can monitor the difference between the two as the wavelength is altered. These instruments usually come with a motor driven mechanism for altering the wavelength, or scanning the sample.