Spectrophotometry is a quantitative measurement of the amount of light that is absorbed by a chemical substance by obtaining the intensity og light that is transmitted through the solution. Spectrophotometric analysis is useful in many areas such as Physics, Chemistry and Molecular Biology. An example of the use of Spectrophotometry is in the measurement of the equilibrium constant of a solution. The concentration of the reactants and products in a solution once it reaches equilibrium is determined using Spectrophotometer.

 

In experiment, four samples with unknown concentrations were given. The goal was to find the concentration of each sample. FIrst, the serial dilution was perfomed on a given stock solution with known concentration. The figure shows the arrangement of the diluted solutions as well as the four unknown samples in a 96-well plate. The absorbance of each sample was obtained from Spectrophotometer at 650nm wavelength. At this wavelength, a linear relationship exists between the concentration and the absorbance of a solution(Beer-Lambert's Law). Using standard curve(relationship between concentration and absorbance) obtained from the diluted solutions, the concentration value of the unknown solutions was extrapolated.