[farah]

I'm curious, why must the concentration of the solution not be too high during wavelenght measurement.
p.s  i'm using cintra 5, a table top spectrometer.

[Nick]

Farah,

In UV/vis absorption spectrophotometry, you measure the amount of light attenuation that occurs as it passes through the sample.  By not appearing at the detector, a photon "reports" the presence of an absorbing molecule.  In order to make a quantitative measurement, there must be a very high probability that only zero or one absorbing molecules reside in the "pathlength" of a single photon.   Why?  Iimagine the case where the concentration is so high, there are several absorbing molecules in the pathlength of a single photon.  Since the photon can only be absorbed once, it's failure to appear at the detector indicates a single absorbing molecule and it cannot report on the many molecules directly behind it.  As a result, the measured concentration is lower than the actual concentration.  This is the basis for the nonlinearity (plateau) observed in a plot of ABS vs. concentration at high concentrations.  To be in the linear regime of Beer's law, one must dilute the sample, ideally below an absorbance of 1.

[farah]

thanx a lot nick. i really appreciated tht