Ph. Eur. 2.2.25. ABSORPTION SPECTROPHOTOMETRY, ULTRAVIOLET AND VISIBLE

European Pharmacopoeia (Ph. Eur.) Chapter 2.2.25, Absorption Spectrophotometry, Ultraviolet and Visible, defines the principles, instrumentation, and qualification requirements for UV–Vis spectrophotometry. This compendial method measures light absorption across UV and visible wavelengths, applying the Beer–Lambert law to confirm identity and quantify concentration. The chapter is harmonized with USP <857> and JP 2.24 under the Pharmacopoeial Discussion Group (PDG).

Principle: Absorbance is proportional to molar absorptivity, path length, and concentration (A = εbc). This linear relationship underpins both identification and quantitative assays.

Instrumentation: UV (deuterium) and visible (tungsten–halogen) light sources; monochromator or suitable wavelength selector; cuvette of defined path length (typically 1 cm); detector; often configured as double-beam spectrophotometers to correct for baseline drift.

Qualification / Performance Verification:

Wavelength accuracy – holmium oxide solution.

Photometric accuracy – potassium dichromate solution.

Stray light – cut-off filters (e.g., potassium chloride).

Resolution – toluene in hexane.
These checks ensure the instrument is fit for compendial use.

Applications:

Identification – compare absorption maxima and spectral profile against reference standards.

Quantification – either compare absorbance to reference standard solutions or use monograph-specific specific absorbance (E1% 1 cm) values.