Basic principles of spectrophotometry:
An absorbance spectrophotometer is an instrument that measures the fraction of the incident light transmitted through a solution. In other words, it is used to measure the amount of light that passes through a sample material and, by comparison to the initial intensity of light reaching the sample, they indirectly measure the amount of light absorbed by that sample.
Spectrophotometers are designed to transmit light of narrow wavelength ranges (see Figure 1 the electromagnetic spectrum). A given compound will not absorb all wavelengths equally-that's why things are different colors (some compounds absorb only wavelengths outside of the visible light spectrum, and that's why there are colorless solutions like water). Because different compounds absorb light at different wavelengths, a spectrophotometer can be used to distinguish compounds by analyzing the pattern of wavelengths absorbed by a given sample. Additionally, the amount of light absorbed is directly proportional to the concentration of absorbing compounds in that sample, so a spectrophotometer can also be used to determine concentrations of compounds in solution. Finally, because particles in suspension will scatter light (thus preventing it from reaching the light detector), spectrophotometers may also be used to estimate the number of cells in suspension.
We will be using a spectrophotometer several times this semester to quantify the concentration of chemicals present in a solution.