Calibration of UV-VIS Spectrophotometer


1.0    Objective

To lay down the procedure for calibration of UV-VIS Spectrophotometer

2.0  Scope

This procedure is applicable for the Calibration of UV-VIS spectrophotometer.

3.0  Responsibilities

QC officer/ Executive : Responsible for the calibration and to review the results of calibration of the instrument.

QC Head/ Manager : Responsible for accountability and effective implementation of the standard procedure for operation of instrument.

4.0  Procedure

4.1  Calibration of instrument is important as :

  • It performs the regular performance checks.
  • It ensures that the testing performed on the respective instrument give the accurate results and meets the standards of GMP and GLP.

4.2 Use the reference standards for calibration along with their certificates.

4.3 Calibration shall be done as follows:

4.3.1 Control of Wavelength: 

  • Chemicals required: 1.4 M perchloric acid, Holmium perchlorate solution.
  1. Preparation of 1 .4 M Perchloric acid preparation : Dilute 11.5 ml of Perchloric acid (70%) to 100 ml with water.
  2. Preparation of Holmium Perchlorate solution:Dissolve about 1.0  gm of Holmium oxide in 1.4 M Perchloric acid with the aid of heating on a  water bath, cool and dilute to 25 ml with same solvent
  • Record the spectrum of Holmium perchlorate solution from 200nm to 600nm using 1.4M Perchloric acid as reference solution. 
  • Note down the maxima observed at wavelength against the acceptance criteria given below:                                              

Wavelength                    Maximum Tolerance

241.15 nm                        240.15 nm  to 242.15 nm

287.15 nm                        286.15 nm to 288.15 nm

361.50 nm                        360.50 nm to 362.50 nm

536.30 nm                        533.30 nm to 539.50 nm

4.3.2 Control of Absorbance:

  • Chemicals required : .005M sulphuric acid,  Potassium dichromate primary standard.

 For Ultra violet region:

  1. Dilute 0.6 ml of Sulphuric acid to 2000 ml with purified water (0.005 M Sulphuric acid).
  2. Weigh accurately 57.0 to 63.0 mg of Potassium dichromate primary standard (previously dried at 130°C for constant weight) and  transfer it to 1000 ml volumetric flask. Dissolve in 0.005M sulphuric acid and make up to the mark with the same acid.
  • Measure the absorbance at 235nm, 257nm, 313nm and 350nm using 0.005M sulphuric acid as reference solution.

 For visible region:

  1. Weigh accurately 57.0 to 63.0 mg of Potassium dichromate primary Standard (previously dried at 130°C for constant weight) and transfer it to 100 ml volumetric flask.
  2. Dissolve in 0.005M sulphuric acid and make up to the mark with the same acid.
  • Measure the absorbance at 430nm using sulphuric acid as reference solution.
  • Calculate the value of A(1% , 1 cm ) using the expression given below :

A (1%, 1 cm) =   Absorbance x 1000 / wt. in g   x  100

  • Check the value of A (1%, 1 cm ) at each wavelength against the acceptance criteria given below :

Wavelength                             Maximum Tolerance

235 nm                                    122.9 nm  to 126.2 nm

257 nm                                    142.8 nm to 146.2 nm

313 nm                                    47.0 nm to 50.3 nm

350 nm                                    105.6 nm to 109.0 nm

430 nm                                    15.7nm  to 16.1nm

4.3.3 Limit of stray light:

  • Prepare a solution of 1.2 % w/v g of Potassium chloride (previously dried at 105°C, 2 hours) Purified water.
  • Determine the absorbance using path length of 1 cm at 200 nm against purified water as blank.
  • Acceptance criteria : Absorbance at 200 nm should be greater than 2.0


4.3.4 Resolution power:

  • Prepare a solution of 0.02 % v/v Toluene in Hexane as follows:
  • Dilute 2.0 ml of toluene to 100.0 ml with hexane. Further dilute 1.0 ml this solution to 100.0 ml with hexane.
  • Record the spectrum of 0.02% v/v solution of toluene in hexane from wavelength 250nm to the wavelength 300 nm.
  • Record the absorbance at the maxima at about 269 nm and minima at about 266
  • Calculate the ratio of absorbance by dividing the absorbance at the maxima at about 269nm and minima at about 266nm
  • Acceptance criteria : The ratio of Absorbance at the maxima at about 269 nm to that  at the minimum at about 266 nm is not less than 1.5.



UV-VIS   :   Ultra violet visible

QC         :   Quality Contro

GMP      :   Good manufacturing practices

GLP       :   Good laboratory practices

nm         :   Nanometer