Question

Spectroscopy measurements may be made in either percent transmittance (% T) or absorbance (A). Based on the mathematical relationship between absorbance and transmittance, A= -log T, explain why calibration curves of absorbance versus concentration may deviate from a straight line when A<0.1 and when A>1.

Answer 1

• unequal light path lengths across the light beam
• unequal absorber concentration across the light beam
• changes in refractive index at high analyte concentration
• shifts in chemical equilibria as a function of concentration
• fluorescence of the sample, in which some of the absorbed light is re-emitted and strikes the detector

Step-by-step explanation:

For a spectroscopic experiment to fulfill the Beer Lambert law, one requirement is that every photon of light must have an equal probability of absorption. Anything that disrupts these conditions leads to a deviation from the law.

The Beer Lambert law is given as follows:

A = Log (I₀/I)

One major source of deviation is stray light. This is any light that has a wave length outside the boundaries.

Answer 2

According to Beer’s Law, A=Ebc, under ideal conditions, a substance’s concentration and its absorbance are directly proportional: a high-concentration solution absorbs more light, and solution of lower concentration absorbs less light. Since concentration and absorbance are proportional, Beer’s Law makes it possible to determine an unknown concentration of phosphate after determining the absorbance.