9/25/23, 2:18 PM SOP for Calibration of UV-Vis Spectrophotometer : Pharmaguideline

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SOP for Calibration of UV-Vis Spectrophotometer

Standard operating procedure to calibrate the UV-Vis Spectrophotometer to verify the performance by Control of Wave length, Control of Absorbance, Limit of Stray Light
and Resolution Power tests.
1.0 OBJECTIVE
To describe the procedure for Calibration of UV-Vis. Spectrophotometer.
2.0 SCOPE
This SOP is applicable for Calibration of UV-Vis. Spectrophotometer.
3.0 RESPONSIBILITY
Officer/ Executive - Quality Control.

4.0 ACCOUNTABILITY
Manager - Quality Control.
5.0 PROCEDURE
5.1 Calibration
5.1.1 Calibration of UV-VIS Spectrophotometer is done in four steps.
A] Control of Wavelength
B] Control of Absorbance
C] Limit of Stray Light
D] Resolution Power
5.1.2 Operate the instruments as per SOP.
5.2 Control of Wavelength
5.2.1 Weight accurately 1.0 gm of Holmium Oxide and dissolve it in 1.4 M Perchloric acid solution. Makeup to 25 ml with the same solvent.
5.2.2 Select the method file of CONTROL OF WAVELENGTH in the instrument.
5.2.3 After selecting the file press Reference button for baseline correction.
5.2.4 Then fill the Cuvette with 1.4M Perchloric acid and put in the sample cubicle and press reference to zero.
5.2.5 After auto zero put the Holmium perchlorate solution in sample cubicle then press start key.
5.2.6 Scan it and verify the wavelength using absorption maxima of Holmium Perchlorate solution. The permitted tolerance is given in below table.
S.No. Maxima Wavelength (nm) Tolerance (nm)
1. 241.15nm 240.15nm to 242.15nm
2. 287.15nm 286.15nm to 288.15nm
3. 361.5nm 360.50nm to 362.50nm
4. 536.3nm 533.30nm to 539.30nm

5.3 Control of Absorbance

5.3.1 Dry a quantity of the Potassium dichromate by heating to constant weight at 130°C.
5.3.2 Weight accurately about 60 mg of dried potassium dichromate and dissolve it in 0.005M sulphuric acid solution. Make up to 1000 ml with the
same solvent. Mark the solution as (A).
.3.3 Weight accurately about 60 mg of dried potassium dichromate and dissolve it in 0.005M sulphuric acid solution. Make up to 100 ml with the
same solvent. Mark the solution as (B).
5.2.7 Select the method file of CONTROL OF ABSORBANCE in the instrument.
5.2.8 After selecting the file press Reference button for baseline correction.
5.2.9 Then fill the Cuvette with 0.005M Sulphuric acid for blank and put in both sample cubicle and press reference to zero.
5.2.10 After auto zero put the Potassium Dichromate solution labeled as solution ‘A’ in sample cubicle then press start key taking absorbance
individually for first four wavelengths mentioned in ‘Table I’.

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9/25/23, 2:18 PM SOP for Calibration of UV-Vis Spectrophotometer : Pharmaguideline

5.2.11 Now take absorbance at 430 nm for solution ‘B’.

5.2.12 Note the absorption maxima of Potassium Dichromate solution at a different wavelength and calculate the absorbance, tolerance is given in
below table.
Table I
S.No. Wavelength (nm) Absorbance E (1%1cm) Maximum tolerance
1. 235 124.5 122.9 to 126.2
2. 257 144.0 142.8 to 145.7
3. 313 48.6 47.0 to 50.3
4. 350 106.6 104.9 to 108.2
5. 430 15.9 15.7 to 16.1

5.4 Limit of Stray light

5.4.1 Dry a quantity of the Potassium chloride by heating to constant weight at 130°C.
5.4.2 Weight accurately 1.20 g of dried potassium chloride and dissolve it in 50 ml distilled water. Make upto 100 ml with the same solvent.
5.4.3 Select the method file of LIMIT OF STRAY LIGHT in the instrument.
5.4.4 After selecting the file press Reference button for baseline correction.
5.4.5 Check the absorbance of above solution using water as a blank at 200 nm.
5.4.6 Absorbance should be greater than 2.0
5.5 Resolution power
5.5.1 Prepare 0.02%v/v solution of Toluene in Hexane UV.
5.5.2 Select the method file of RESOLUTION POWER in the instrument.
5.5.3 After selecting the file press Reference button for baseline correction.
5.5.4 Measure the absorbance of above solution at 266 nm and 269 nm using Hexane UV as blank solution.
5.5.5 The ratio of absorbance maxima at 269 nm to that of 266 nm minima should be more than 1.5
5.5.6 Note down the report in the internal calibration certificate and in Instrument Logbook.
6.0 ABBREVIATIONS
6.1 SOP - Standard Operating Procedure
6.2 nm - Nanometer
6.3 VIS - Visible
6.4 UV - Ultra Violet

Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of pharmaguideline.com, a widely-read pharmaceutical
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