Question

5) What is the absorbance at 280 nm of a 3 µL of 1µM protein solution with a molar absorptivity of 23,000 cm-1 M-1 measured in a 0.1cm path length cell? Explain the molecular basis for this phenomena.

Answer 1

A/C to Beer-Lambert Law

where,

A= absorbance, C= concentration of solution , l = length of solution from which light passes through

A= 2300 /cm.M x 0.1 cm x 1x 10^-6 M = 0.23 x10^-3

Beer’s law relates absorbance to both transmittance and to the concentration of the absorbing species.

A = –logT = εlC

In Molecular absorption a hollow cathode lamp provides the necessary line source of radiation. After absorbing

radiation a molecule become excited it moves from a lower energy state to a higher energy state. In returning to the lower energy state the molecule may emit radiation. Spectrophotometry work on this principle.