Spectrophotometers measure the amount of light absorbed by a sample at a particular wavelength. The absorbance of the sample depends on the electronic structures of the molecules present in the sample. Measurements are usually made at a wavelength that is close to the absorbance maximum for the molecule of interest in the sample.
The diagram below shows the elements present in a typical spectrophotometer. The light sources used in most spectrophotometers emit either ultraviolet or visible light. Light
(Io) passes from a source to a monochromator, which can be adjusted to allow only light of a defined wavelength to pass through. The monochromatic (I) light then passes through a cuvette containing the sample to a detector.
The spectrophotometer compares the fraction of light passing through the monochromator (I0) to the light reaching the detector (I) and computes the transmittance (T) as I/I0. Absorbance (A) is a logarithmic function of the transmittance and is calculated as:
A = log10(1/T) = log10(I0/I)
Spectrophotometers can express data as either % transmittance or absorbance. Most investigators prefer to collect absorbance values, because the absorbance of a compound is directly proportional to its concentration. Recall the Lambert-Beer Law, traditionally expressed as:
A =\(\varepsilon\)b C
where \(\varepsilon\) is the molar extinction coefficient of a compound, b is the length of the light path through the sample, and C is the molar concentration of the compound. Cuvettes are formulated to have a 1 cm light path, and the molar extinction coefficient is expressed as L/moles-cm. Consequently, absorbance is a unitless value.