Controle de qualidade

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 spectrum in 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 measuring the 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 usually measured 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 light intensity (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 the absorbance 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 the solution 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.
1cm