How to Calibrate UV/Visible Spectrophotometer

Table of Contents

• Calibration description
  • Reagents and instruments
  • Objective
  • Scope
  • Responsibility
• Frequency
• Calibration parameters
• Control of wavelength procedure
• Control of absorbance procedure
• Limit of Stray Light
• Resolution Power
• Line Flatness

Calibration Description

Reagents and chemicals

• Perchloric acid
• Holmium oxide
• Sulfuric acid
• Potassium dichromate
• Potassium chloride
• Toluene
• Hexane
• Distilled water

Objective

The purpose of this SOP is to detail the procedure for the calibration of the UV spectrophotometer.

Scope

This procedure applies to all UV spectrophotometers of all the department (QC or ARD) of the company.

Responsibility

Research Scientists or chemists or analyst responsible for the calibration of the UV spectrophotometer

HOD (Head of the department)

To ensure the implementation of this SOP in the respective departments of the company (as applicable)

Frequency for calibration of UV

Once in three months ± 5 days

Calibration parameters

The following parameters are performed during UV calibration

• Control of Wavelengths
• Control of absorbance
• Limit of stray light
• Resolution power
• Line Flatness

Control of Wavelengths

Solution preparation

1.4 M perchloric acid solution

Pipette out 11.5 ml of Perchloric acid (about 60%) and transfer it into a 100 ml of volumetric flask. Make up the volume with water.

Note: Perchloric acid of higher strength/concertation can also be used for the preparation of 1.4M Perchloric acid but dilution should be adjusted accordingly

Holmium perchlorate solution

Weigh accurately about 400mg of Holmium oxide and transfer it into a 10 ml volumetric flask containing 1.4M Perchloric acid solution. Shake the solution well to dissolve Holmium oxide and make the volume to the mark with 1.4M Perchloric acid solution.

Procedure

• Take a pair of cuvettes having length of 1cm and perform the baseline correction using 1.4M Perchloric solution from 200 nm to 600 nm
• Remove the cell from the sample compartment
• Rinse the cell with Holmium perchlorate solution and fill the cell with the same. Clean the outer surface of the cell with tissue paper. Place the cell in the sample compartment and record the spectra

Acceptance criteria

The permitted tolerance is ±1nm for the range of 200 nm to 400 nm and ± 3 for the range of 400 nm to 600 nm

Table-1

Control of absorbance procedure

0.005 M Sulfuric acid Solution preparation

Dilute 0.41 ml of Sulfuric acid with 1500 ml of water

Solution A

Weigh accurately the quantity NLT 57 mg and NMT 63 mg of Potassium dichromate standards and transfer it into a 100 ml of volumetric flask containing 50 ml of 0.005M Sulfuric acid. Shake well to dissolve the Potassium dichromate completely. Make up the volume to mark with 0.005M Sulfuric acid solution.

Solution B

Weigh accurately the quantity NLT 57 mg and NMT 63 mg of Potassium dichromate standards and transfer it into a 100 ml of volumetric flask containing 50 ml of 0.005M Sulfuric acid. Shake well to dissolve the Potassium dichromate completely. Make up the volume to mark with 0.005M Sulfuric acid solution.

Procedure

• Take a pair of cuvettes having length of 1 cm
• Perform the baseline correction using 0.005 M Sulfuric acid solution from 200 nm to 600 nm
• Remove the cell from the sample compartment
• Rinse the cell with solution A and fill the cell with the same. Clean the outer surface of the cell with tissue paper. Place the cell in the sample compartment and record the spectra between 200 nm to 600 nm and measure the absorbance at 235 nm, 257 nm , 313 nm and 350 nm
• Rinse the cell with solution B and fill the cell with the same. Clean the outer surface of the cell with tissue paper. Place the cell in the sample compartment and measured the absorbance at 430 nm
• Calculate the specific absorbance using the following equation:

Specific Absorbance at “A” nm=Absorance at “A” nm × final volume×10Weight of the sample in mg

Acceptance criteria: 

The specific absorbance against each wavelength should be as follows (table-2):

Table-2

Limit of Stray Light

Solution preparation

Prepare 1.2% w/v of Potassium chloride solution in distilled wetter ( Dilute 1.2 gram of Potassium chloride to 100 ml with water. Shake to dissolve)

Procedure

• Take a pair of cuvette having a length of 1cm
• Perform autozero with distilled water
• Remove the cell from the sample compartment
• Rinse the cell with Potassium chloride solution and fill the cell with the same, and place the cell in the sample compartment
• Measure the absorbance at 200 nm

Acceptance criteria

The specific absorbance should not be less than 2

Resolution power

0.02% v/v Toluene in Hexane solution preparation

Pipette out 2 ml of Toluene and transfer into 200 ml volumetric flask and make up the volume with Hexane. Further, pipette out 2 ml of resulting solution and transfer into 100 ml volumetric flask and make up the volume to mark with Hexane

procedure

Record the spectra in the range of 260 nm to 275 nm using Hexane in the reference cell

Calculation

Ratio of the Absorbance=Absorbance at 269nmAbsorbance at 266nm

Acceptance criteria

The ratio of absorbance should not be less than 1.5

Line Flatness

Procedure

• Perform the test with an Air blank in the sample and in the reference compartment
• Scan in the wavelength range of 220 nm to 700 nm and generate the spectraa in the % transmittance mode

Acceptance criteria

The line obtained should not deviate from the horizontal line at any point by more than 90% and less than 102% transmittance

Abbreviations:

• HOD: Head of department
• Std: Standard
• SOP: Standard operating procedure
• COA: Certificate of analysis
• UV: Ultraviolet
• nm: Nanometer
• NLT: Not less than
• NMT: Not more than

References:

• EP
• USP