Effect of high optical densities on the Beer-Lambert-Bouguer law

Effect of high optical densities on the Beer-Lambert-Bouguer law 

The Beer-Lambert-Bouguer law is no more respected for two reasons, one is due to the instrument and the other depends on the chromophore itself 

The linearity between the optical density and the concentration of a chromophore is no more respected at high optical densities. This can be the result of the saturation of the photomultiplier which will be not able to count more than a precise number of photons. When this number is reached, the optical density given by the spectrophotometer will be the highest and we are going to say that the photomultiplier is saturated. Beyond the concentration of saturation the spectrophotometer will always give the same optical density that corresponds to that of saturation. 

The second reason is owed to the chromophore itself. At high concentrations, aggregation could occur. In this case, we are going to have two values of c, corresponding to low and high concentrations. Plotting the optical density as a function of the macromolecule concentration will yield two plots with two different values of c. 

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