This document discusses various deviations from Beer's Law that can occur when using UV-Vis spectroscopy. There are three main types of deviations: 1) True deviations due to high analyte concentrations that cause interactions between molecules and changes in optical properties. 2) Instrumental deviations caused by factors like use of polychromatic light, improper slit width, stray light, mismatched cells, and scanning speed. 3) Chemical deviations due to shifts in chemical equilibria with changing concentration, like for pH indicators. Nonlinearity can be minimized by optimizing factors like concentration, slit width, and wavelength range used.

1. Deviations from Beers Lamberts law
Dr. Vrushali Tambe
PES Modern College of Pharmacy
(For ladies)

2. Deviations from Beers Lamberts law
1. True/ real/ fundamental deviation
2. Instrumental deviations
3. Chemical deviation

3. True deviation
•Deviations in absorptivity coefficients at high concentrations
(>0.01M) due to electrostatic interactions between molecules in
close proximity
•scattering of light due to particulates in the sample
•fluorescence or phosphorescence of the sample
•changes in refractive index at high analyte concentration

4. Changes in the solution refractive index with analyte
concentration can also cause nonlinearity by altering
the position, size, or solid angle of the image
transmitted to the detector, the reflectance loss at the
cell wall-solution interface of the analyte molar
absorptivity.
In the last case, epsilon in Beer's law is replaced by

5. • Nonlinearity can occur if the analyte molar absorptivity
is dependent on the analyte concentration.
• Such effects are usually minor and occur at relatively
high concentrations (> 10-2 M).
• Differences in solute-solvent interactions, solute-solute
interactions, or hydrogen bonding at high
concentrations can change the chemical or electrostatic
environment and hence the absorptivity of the analyte.
• SPEED OF LIGHT IS REDUCED AT HIGHER
CONCENTRATION
• RI of sample should be less than 1 (10-6 to 10-3 M)

6. CHEMICAL DEVIATION
Shifts in chemical
equilibria as a function of
concentration
• Equilibrium shift
– pH indicators

7. Phenol red

8. INSTRUMENTAL DEVIATIONS
• Polychromatic Light
– More than one
wavelength
Non-monochromatic radiation,
deviations can be minimized by using
a relatively flat part of the absorption
spectrum such as the maximum of an
absorption band

9. Slit width
• Spectral band width (the width, at half the maximum intensity, of
the band of light leaving the monochromator)
• Natural band width (the width of the sample absorption band at
half the absorption maximum)
• SPW should be 1/10th of NBW. If NBW is 200nm, than SPW should be 20nm
SPW= Slit width* dispersion
of monochromator

10. Stray light
• Any radiation reaching detector other than
the narrow range of wavelengths normally
transmitted by monochromator.
• Arises from scattering and refraction inside
the monochromator, due to imperfections on
the optical surfaces.
• More evident at high absorbance value.
• Aobs = log Io + SL
It + SL

12. Mismatched Cells or Cuvettes
If the cells holding the analyte and the blank
solutions are having different path-lengths, or
unequal optical characteristics, there would be a
deviation observed in Beer- Lambert law.

13. Scanning Speed
• At higher scanning speed their
will be electrical and mechanical
dumping of recorder
• Scan is shifted towards the
direction of recording
• λmax will decrease
• Absorbance at λmax will
decrease
• Absorbance at λmax will
increase
• Resolution will decrease