Quantification of Proteins in Solution by Spectrophotometer

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Experiment 2

Quantification of Proteins in Solution by Spectrophotometer

Lab bench# 1

Absorption spectroscopy is a common method for finding the concentration of proteins or protein complexes in a solution. Proteins absorb light at specific wavelengths and can be defined by the equation A = log (Io/I). This equation states that an absorbance at a specific wavelength, A is equal to the log of the ratio of incident light intensity (Io), to transmitted light intensity (I). A spectrophotometer can be used quantitatively and qualitatively. A spectrophotometer is used qualitatively to obtain an absorption spectrum, which can be obtained by plotting the absorbance values, over the range of wavelengths tested for the
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It is best to consider wavelength of maximum absorption because stronger the intensity, the more accurate will be the readings for absorbance.
As seen from table 1, the path lengths remain the same as the cuvettes used were of the same size. The Beer-Lambert Law states that Abs = Ɛ.c.l, where Ɛ = molar extinction coefficient, c = concentration of protein solution, and l = path length of light through medium. Thus, it is noted that absorbance and path length share a directly proportional relationship, i.e. if path length increases, absorbance increases as well. It was clearly observed in the wide and narrow test-tubes, that as the path length was doubled, the absorbance value doubled too (Srivastava, 2008).
Also, from the same equation, it can be determined that absorbance and concentration share a directly proportional relationship meaning that as the concentration decreases, it directly affects the absorbance value obtained, and this value decreases too. Thus, as seen for the four cuvettes tested (in Table 1) as the concentration is halved in every cuvette, the absorbance value is halved correspondingly as well.
It is known that the Beer-Lambert law says absorbance is proportional to number of absorbing molecules, and that this is valid for a variety of compounds over a wide range of concentrations. But even as the molar extinction coefficient is seen to be attributed to wavelength, it is true only for monochromatic