Hi,

In HPLC-UV - has anyone got an explanation why there is a negative deviation of Beer's law when concentration in flow cell becomes too high?? I use a small volume detector cell (1 ul, 5 mm pathlength) to limit X-band spreading. Any references??

Many thanks

Because Beer's law applies to dilute solutions ;-)

Stray light will limit the absorbance any detector can measure. You will see non-linearity if you start to approach the sray light imposed absorbance limit normally, >1.5 AU.

Actual Beer's law deviations occur at high concentration because the analyte itself starts to affect the environment the analyte is in.

Also wierd things like concentration dependent dimerization or photodegradation can affect the linearity.

Real data like the concentrations, absorbances, analytes, and wavelengths are more likely to yield real help. Good luck

I think this is a problem with small volume flow cells that have also a smaller path length. The concentration of substance in the detector cell has to be twice that in a standard 1cm detector cell to generate the same absorbance. Therefore you can run into linearity problems more easily.