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B. GENERAL TESTS

Spectrophotometry

Spectrophotometry is designed to measure the degree of absorption of light by a
substance, in a definite and narrow wavelength range. The absorption spectrumin
the visible and ultraviolet regions of a substance in a solution is characteristic
depending on its chemical structure. Therefore, Spectrophotometry is used to identify
a substance by measuringthe absorbances at various wavelengths. This method is
applicable to identification tests, purity tests, and assays, in which the absorbance of
a solution with a certain concentration is usuallymeasured at the wavelength of the
maximum absorption (λmax.) or the minimum absorption (λmin.).
When monochromatic light passes through a substance in a solution, the ratio of
the transmitted lightintensity (I) to the incident light intensity (I0) is called
transmittance (T), while the common logarithm of the reciprocal of transmittance is
called absorbance (A).
T=

I
I0

A = log

Io= − log T
I

The absorbance (A) is proportional to the concentration (c) of the solution and the
length (l) of the layer of solution through which the light passes.

A = kcl ( k = cons tan t )Calculated on the basis that l

(

)

called specific absorbance E1% ;
1cm

is 1 cm and c is 1% w/v solution, the absorbance is
calculated on the basis that l is 1 cm and c is 1

mol/l,the absorbance is called molecular extinction coefficient (E).
The molecular extinction coefficient at the wavelength of the maximum of
absorption is expressed as Emax.
The measurement of theabsorbance is performed for solutions using the
specified solvent.
The appropriate concentration of a solution is one in which the measured
absorbance ranges between 0.2 and 0.7. If the absorbance of thesolution indicates a
higher value than described above, dilute the solution with the solvent to a suitable
concentration, and measure again.
Use the formulas, given below, to obtain E1% or E....