Uv Calibration

In: Science

Submitted By Siddheshswami
Words 982
Pages 4
UV-Visible Spectrophotometer
Dept. of Analytical chemistry
Razi vaccine serum research institute

The primary objective of GLP is to ensure the generation of high quality data. Essential to this is the ability to produce estimates for the measurement uncertainty associated with calibrations.


The spectrophotometer has become the workhorse for high throughput routine testing for a wide variety of analysts in chemical and biochemical analysis laboratories. It is important for accuracy and reproducibility in measurement that this instrument is correctly set up and calibrated.

Increasing Wavelength









400 cm-1




25 000 nm

Increasing Energy

K-shell electrons Outer-shell electrons Molecular vibrations Molecular

Conventional Spectrophotometer:

The factors that must be calibrated:
Control of wavelengths
Control of absorbance
Limit of stray light
Spectral slit width

Control of wavelengths:
Wavelength standards:
The ideal wavelength standard would have very narrow and very well defined peaks.

Control of wavelengths:
•Solution of holmium(III) in perchloric acid: 240–
640 nm.
•Holmium oxide filters: 280–640 nm.
•Didymium filters: 400–750 nm.
•Using of spectral lines in low-pressure discharge lamps: 656.1 nm produced by the D2 lamp.

Holmium(III) in perchloric acid:
Consists of a 4% W/V solution of Holmium Oxide in
1.4M Perchloric Acid. λmax………………241.1 ± 0.4nm
278.0 ± 0.4nm
287.5 ± 0.4nm
361.2 ± 0.4nm
416.6 ± 0.4nm
451.3 ± 0.4nm
458.3 ± 0.4nm
537.5 ± 0.4nm
640.8 ± 0.4nm

Holmium oxide filters:

Didymium filters:

Discharge lamps:
A quick check of the wavelength accuracy of a…...

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