What factors contribute to deviations from the Beer-Lambert’s Absorbance law?

 deviations from the Beer-Lambert’s Absorbance law?

Deviations from Beer- Lambert’s law

Beer–Lambert’s Absorbance law is a universally accepted relationship which helps calculation of concentration of an absorbing species from measured absorbance values. Under ideal conditions absorbance versus concentration plot is a straight line passing through the origin. However, under real situations a curvature in the plot is observed beyond a particular concentration. The curvature can be both positive or negative depending on the nature of absorbing species and experimental conditions. Deviations from the law were discussed in an earlier article. The present article will discuss the reasons responsible for such deviations.

Positive deviations are observed when the measured absorbance values are more than the expected theoretical values and negative deviations result when the measured values are lower than the theoretical values.

Limitations of the Law

Beer – Lambert’s law generally behaves ideally for concentrations below a critical limit. For most absorbing molecules non-linear behaviour is observed at concentrations above 10mM. However, some absorbing molecules such as methylene blue tend to exhibit deviations from linearity at concentrations as low as 10 µM. Solute – solvent interactions at higher concentrations and even hydrogen bonding are responsible for such deviations..

Chemical factors

Change in pH

Some absorbing molecules undergo change in colour with change in pH of the solvent medium. An example is Phenol red which changes colour from yellow in acidic media to red in basic media due to internal migration of proton accompanied by a switch between a single and an adjacent double bond.

Another example is aqueous solution of potassium dichromate which changes from yellow to orange on increasing the concentration of hydrogen ions due to chromate – dichromate conversion

Double exponent: use braces to clarify         Undefined control sequence \rightleftarrows            Double exponent: use braces to clarify
Chromate                                 Dichromate
(Yellow)                                 (Orange)

Complexation , Dissociation or Association

Complex formation due to association can result in colour changes with concentration changes. An example is cobalt chloride which changes colour from green to blue due to association

2CoCl2             Undefined control sequence \rightleftarrows         Co(CoCl4)
(Pink)                                   (Blue)

The degree of association increases with increase in concentration

Instrumental factors

Wavelength selection

Beer- Lambert’s law holds true strictly for single wavelength light. However, due to inefficiencies of monochromator a range of wavelengths gets isolated. In addition stray light also results from internal reflections from optical components such as gratings, lenses and optical windows. Absorption resulting from stray light contributions will show deviations from the Beer- Lambert’s law

Mismatch of solution and cells

Sample and reference blank solutions composition variations or optical mismatch of windows of cuvettes will result in deviations from Beer- Lambert’s law.

To minimize the deviations the following precautions should be taken while reading absorbance of solutions to provide high accuracy of results:

• Always use optically matched pair of measuring cells
• The concentration of analyte should be below the linear range of Beer -Lambert’s plot.
• pH should be maintained at specified value for both blank and sample solutions
• The composition of blank and sample solution should match as closely as possible