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Calibration of UV-Visible spectrophotometer

In Pharmaceutical UV-Visible spectrophotometer is widely used for analytical purposes in the Quality control department. So to maintain its accuracy and working conditions, calibration of UV-Visible spectrophotometer is performed timely.

Calibration of UV-Visible spectrophotometer by parts

Check the following During calibration of UV-Visible spectrophotometer

Calibration of UV-Visible spectrophotometer
  • Absorption cells
  • Control of absorbance
  • Photo metric linearity
  • Limit of stray light
  • Resolution power
  • Control of Wavelengths

Absorption cells

Fill the cuvette with milli-Q water at 240 nm, and absorption is not greater than 0.093 of the individual cuvette.

Rotate both cuvettes (180°) one by one.

Acceptance criteria: On rotation of cuvette should give absorbance difference not more than 0.005.

Control of absorbance:

For UV region:

Prepared 0.005M sulfuric acid:

Weigh accurately 60.0 mg of potassium dichromate, which previously dried to constant weight at 130°C in 1000.0ml and dissolved in 0.005 M sulphuric acid.

Measure the absorbance at 235nm, 257 nm, 313nm, and 350 nm using 0.005 M H2SO4 as blank.

For visible region:

Weight about 60.0mg of potassium dichromate, which previously dried to constant weight at 130°C and transferred to 100.0 ml volumetric flask, dissolved in 0.005 M sulphuric acid.

Measure the absorbance at 430 nm using 0.005M sulphuric acid as blank.

Photometric Linearity:

Acceptance criteria

wavelengths
(nm)
Specific
abs.
Max.
Tolerance
235 124.5122.9 to 126.2
257 144.5142.8 to 146.2
313 48.647.0 to 50.3
350 107.3105.6 to 109.0

Photometric Linearity:

Weight 50 mg Potassium dichromate and dissolved in 0.005 M Sulphuric acid in a 50 ml volumetric flask.

Further, dilute the above solution in the following way

1 ml ≥ 100 ml

2 ml ≥ 100 ml

3 ml ≥ 100 ml

4 ml ≥ 100 ml

5 ml ≥ 100 ml

Take consecutive three readings of each dilution at wavelength 235,257, 313, and 350 nm.

Acceptance criteria:

At each wavelength mean value ≥of 0.999

Limit of stray light

Record absorbance of 1.2% solution of Potassium chloride in water at 200nm using water as blank

Acceptance criteria: Absorbance should be greater than 2.0

Resolution power

Record the spectrum of 0.02% v/v solution of toluene in hexane at 250nm to 300 nm.

Acceptance criteria

The absorbance maximum at 269 nm to the minimum at about 266 nm should not be less than 1.5.

Control of Wavelengths

Dissolved 1.0 g of holmium oxide in .1.4M perchloric acid to 25ml to prepare 4.0% w/v solution of holmium oxide.

Record spectrum of holmium perchlorate solution from 200 nm to 600 nm using 1.4 M perchloric acid as a reference solution.

Acceptance criteria

WavelengthMaxi. Tolerance
241.14 240.15 to 242.15
287.15 286.15 to 288.15
361.50 360.50 to 362.50
536.30 533.30 to 539.30

Out of Calibration

Suppose the instrument or any parts of it are found out of calibration. Immediately Stopped any activity and Labeled the instruments “Out of Calibration.” Follow the SOP for “Handling of out of calibration.”

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