Internal calibration of PNP

The purpose of this experiment is to determine up to which PNP concentration, the Beer-Lambert-Bouguer law is still linear. In fact, this law cannot be applied when the optical saturation of the photomultiplier is reached and/or when the chromophore aggregates. Each spectrophotometer cannot count more than a precise number of photons. When this number is reached, the OD given by the spectrophotometer will be the highest, and the photomultiplier is saturated. Beyond the saturation concentration, the spectrophotometer will always give the same OD that corresponds to that of saturation. 

Measure the ODs at 405 nm and 430 nm vs. the blank solution where PNP is replaced by water. 

Plot the OD as a function of PNP concentration in the tube at the two wavelengths and determine for each wavelength the maximum PNP concentration at which the Beer-Lambert-Bouguer law is still linear. 

Plots obtained show that above 0.07 mM, OD continues to increase, indicating that PNP is probably aggregating. The aggregate does not have the same absorbance properties as the monomer. 

In order to perform qualitative and quantitative studies with /3-galactosidase, we should work in experimental conditions such that only the monomer of PNP is detected. Also, one 

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