Spectrophotometers contain a white light source, a diffraction grating, and a slit to select a specific wavelength. The selected wavelength of light passes through a sample, and any light that is absorbed is quantified as the absorbance. The absorbance range is 0 (no light absorbed) to 1 or more (lots of light absorbed). According to Beer’s law, absorbance and concentration are directionally proportional: samples with a high concentration of light absorbing particles have a high absorbance, and those with a low concentration of light absorbing particles have a low absorbance. In principle, any difference in absorbance can be measured with the spectrophotometer, but in practice, it is impossible to tell the difference between two samples if they are (1) too optically dense, meaning basically no light gets through, (2) too dilute, meaning practically all of the light gets through, giving both samples an absorbance of nearly 0, or (3) too similar, meaning there are not enough significant figures.

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