QUANTIFICATION OF THE ACTIVE PRINCIPLE BY THE SPECTROPHOTOMETRIC METHOD

The spectrophotometric method is commonly used in the pharmacopoeia and generally in the pharmaceutical industry. Many active principles found in drugs have chemical groups in their structure that absorb UV-light and and can therefore be quantified.

Quantum theory defines the total energy of a molecule as the sum of three energies:

• Electronic energy: Ee
• Vibrational energy: Ev        Et = Ee + Ev + Er
• Rotational energy: Er

UV absorption depends essentially on Ee. The relevant region of the electromagnetic spectrum ranges from 10 to 380 nanometers. In reality, conventional instruments explore the region between 200 and 380 nm, because below 180 nm, the radiation is absorbed by the air and the measurement needs to be taken in a vacuum.

Applications:

• Qualitative: useful for identification (by comparison with standards); however, UV/Visible absorption is not sufficient to identify a substance alone, but can sometimes usefully complement infrared, mass spectrometry, NMR and other spectral methods.

• Quantitative: useful for assays; it is a fast and precise method, based on the exploitation of the Beer-Lambert law that links absorption to the concentration of the molecules in solution, for a given wavelength λ.