Spectrophotometry
Spectrophotometry is a method to measure how much a chemical substance absorbs light by measuring
the intensity of light as a beam of light passes through sample solution. The basic principle is that each
compound absorbs or transmits light over a certain range of wavelength.
The absorption spectroscopy has been widely used for monitoring the rate of chemical reactions. During
the reaction, if there is either appearance of colour in a colourless solution or disappearance of colour in
a coloured solution or a species which absorbed at a specific wavelength is formed, the spectroscopic
technique can be used. Instruments like colorimeters and spectrophotometers are available to cover the
visible, near infrared and ultra violet region of the spectrum (200-1000 nm). The absorption
spectroscopy is governed by well-known Beer-Lambert’s Law according to which:
Absorbance or optical density = I0 /It = Ecl
where I0 and It are the intensity of incident and transmitted light, respectively, c is concentration of
solution, l is the path length of beam of light within the solution and E is molar extinction coefficient
which is characteristic of the absorbing substance. The instruments give the optical density directly. If l
remains constant
Optical density = Constant × c
or Absorbance ∝ c
First the Beer-Lambert Law is tested with the solutions of known composition at λmax of solution by
plotting (absorbance) versus concentration . The concentration range for which the plot is a straight line
is determined and method is applied within this concentration range.
The optical density of samples are measured at different times using same cell (to keep l constant) and
concentration can be calculated using relation c = A/El. To verify the order of reaction, optical density
data can be used directly. If the species is a reactant, then its absorption will decrease as time goes by. If
the species is a product, then its absorption will increase as time goes by.Kinetics is the study of how
concentrations of reactants and/or products change as time goes by.