Experiment 2: Quantification of Proteins in a Solution by Spectrophotometry
Molecules of different substances are unique in many different ways through size, shape, polarity and the type/amount of light they can absorb or choose not to absorb. These characteristics, like the efficiency of absorbing light cannot be differentiated with our naked eye, including the detecting of the extinction co-efficient. Spectrophotometry is of importance because it is a technique used with a machine that allows scientists to identify the type of compound in a solution and the amount. This is of importance to chemistry, biochemistry, physics and the medical field. For example one could determine the seriousness of a heart attack by taking an enzyme from the blood of a patient and comparing it to a standard curve, determining where the range remains constant.
The spectrophotometer contains a light detector that replaces our eye and the source of light is made to produce various wavelengths. With absorbance values in a compound compared to wavelengths an absorption spectrum can be made (Department of Biology, 2013).With this spectrum we are able to differentiate molecules from their unique absorption patterns and determine the maximum wavelength the compound absorbs at. The amount of light a solution absorbs is proportional to the concentration of molecules in a solution as well as the distance light has to travel through, this is known as the Beer-Lambert Law and can be represented as Log [Io/ I] = absorbance = E c l (Horton et al., 2006). E is the extinction co-efficient, c is the concentration, and l is the pathlength of light through the medium. The wavelength that determines E is where the compounds absorb at its maximum. Once the extinction co-efficient is known, solutions of the same compound of an unknown concentration can be quantified (Department of Biology, 2013).
The Beer-Lambert law only works for a small range of concentrations, so before using...