Spectrophotometry is a method to measure how much a substance absorbs light by measuring the intensity of light as a beam of light passes through sample solution. The basic principle is that each compound absorbs or transmits light over a certain range of wavelength. This measurement can also be used to measure the amount of a known chemical substance. Spectrophotometry is one of the most useful methods of quantitative analysis in various fields such as chemistry, physics, biochemistry, material and chemical engineering and clinical applications. Spectrophotometry is widely used for quantitative analysis in various areas (e.g., chemistry, physics, biology, biochemistry, material and chemical engineering, clinical applications, industrial applications, etc).
A spectrophotometer is an instrument that measures the amount of photons (the intensity of light) absorbed after it passes through sample solution. A spectrophotometer, in general, consists of two devices; a spectrometer and a photometer. A spectrometer is a device that produces, typically disperses and measures light. A photometer indicates the photoelectric detector that measures the intensity of light. With the spectrophotometer, the amount of a known chemical substance (concentrations) can also be determined by measuring the intensity of light detected.
Depending on the range of wavelength of light source, it can be classified into two different types
UV-visible spectrophotometer: uses light over the ultraviolet range (185 - 400 nm) and visible range (400 - 700 nm) of electromagnetic radiation spectrum.
IR spectrophotometer: uses light over the infrared range (700 - 15000 nm) of electromagnetic radiation spectrum.
In visible spectrophotometry, the absorption or the transmission of a certain substance can be determined by the observed color. For instance, a solution sample that absorbs light over all visible ranges (i.e., transmits none of visible wavelengths) appears black in theory. On the other hand, if all visible wavelengths are transmitted (i.e., absorbs nothing), the solution sample appears white. If a solution sample absorbs red light (~700 nm), it appears green because green is the complementary color of red. Visible spectrophotometers, in practice, use a prism to narrow down a certain range of wavelength (to filter out other wavelengths) so that the particular beam of light is passed through a solution sample
Beer-Lambert Law (also known as Beer's Law) states that there is a linear relationship between the absorbance and the concentration of a sample. For this reason, Beer's Law can only be applied when there is a linear relationship.