Principle and Calibration of Ultraviolet and Visible Absorption Spectrophotometry : Pharmaceutical Guidelines

Principle and Calibration of Ultraviolet and Visible Absorption Spectrophotometry

Everything about principle and calibration of uv spectrophotometer, Control of wavelengths, Control of absorbance, Limit of stray light and Resolution power

Ultraviolet and visible absorption spectrophotometry is the measurement of the absorption of monochromatic radiation by solutions of chemical substances, in the range of 185 nm to 380 nm, and 380 nm to 780 nm of the spectrum, respectively.
The magnitude of the absorption of a solution is expressed in terms of the absorbance, A, defined as the logarithm to base 10 of the reciprocal of transmittance (T) for monochromatic radiation:
For convenience of reference and for ease in calculations, the specific absorbance of a 1 per cent w/v solution is adopted in this Pharmacopoeia for several substances unless otherwise indicated, and it refers to the absorbance of a 1 per cent w/v solution in a 1 cm cell and measured at a defined wavelength.
It is evaluated by the expression
A(l percent, 1 cm) =A/cl,
where c is the concentration of the absorbing substance expressed as percentage w/v and I is the thickness of the absorbing layer in cm. The value of A (1 per cent, 1 cm) at a particular wavelength in a given solvent is a property of the absorbing substance.
Unless otherwise stated, measure the absorbance at the prescribed wavelength using a path length of 1 cm and at 24° to 26°. Unless otherwise stated, the measurements are carried out with reference to the same solvent or the same mixture of solvents.


A spectrophotometer, suitable for measuring in the ultraviolet and visible ranges of the spectrum consists of an optical system capable of producing monochromatic light in the range of 200 nm to 800 nm and a device suitable for measuring the absorbance.
The two empty cells used for the solutions under examination and the reference liquid must have the same spectral characteristics. Where double-beam-recording instruments are used, the solvent cell is placed in the reference beam.

Related: SOP for Analysis on UV- Visible Spectrophotometer

Control of wavelengths

Verify the wavelength scale using the absorption maxima of holmium perchlorate solution, the line of hydrogen or deuterium discharge lamp or the lines of a mercury vapour are shown below. The permitted tolerance is ± 1nm for the range 200 nm to 400 nm and ± 3 nm for the range 400 nm to 600 nm

Control of absorbance

Check the absorbance using suitable filters or a solution of potassium dichromate UV at the wavelengths indicated in Table 1, which gives for each wavelength the exact values and permitted limits of the specific absorbance. The tolerance for the absorbance is ± 0.01.
Use solutions of potassium dichromate UV which has been previously dried to constant weight at 130°. For the control of absorbance at 235 nm, 257 nm, 313 nm and 350 nm, dissolve 57.0-63.0 mg of potassium dichromate UV in 0.005 M sulphuric acid and dilute to 1000.0 ml with the same acid. For the control of absorbance at 430 nm, dissolve 57.0-63.0 mg of potassium dichromate UV in 0.005 M sulphuric acid and dilute to 100.0 ml with the same acid.

Limit of stray light

Stray light may be detected at a given wavelength with suitable filters or solutions; for example, absorbance of a 1.2 per cent w/v solution of potassium chloride in a 1cm cell should be greater than 2.0 at about 200 nm when compared with water as reference liquid.

Resolution power

When stated in a monograph, record the spectrum of 0.02 per cent v/v solution of toluene in hexane.

Also see: Calibration of UV / Visible Spectrophotometer

Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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