SOP for Calibration of Shimadzu UV–1800 UV Spectrophotometer

OBJECTIVE

To lay down a procedure for the Calibration of the UV Spectrophotometer (UV 1800) using UV Probe software. 

SCOPE

This applies to the UV spectroscopy calibration using UV probe software for Shimadzu UV 1800. 

RESPONSIBILITY

QC Executives are responsible for calibrating once every six months & after every repair or service. 

ACCOUNTABILITY

Assistant Manager – Quality Control 

Manager – Quality Assurance 

CALIBRATION PROCEDURE

Calibration of the UV-VIS Spectrophotometer is done in the following steps.

1. Control of Wavelength 
2. Control of Absorbance 
3. Limit of Stray Light 
4. Resolution Power 
5. Photometric Linearity 
6. Baseline Flatness 
7. Specification of Cells 

ALSO READ: SOP for Operation of Shimadzu UV–1800 UV Spectrophotometer

Control of Wavelength: 

• Preparation of 1.4 M Perchloric acid solution: Take 11.5 mL of Perchloric acid into a clean 100 mL volumetric flask. Dissolve & dilute to the volume with water Dilute 8.5 mL to 100 mL with water in case of 70% Perchloric acid.
• Preparation of Holmium Perchlorate: Weigh accurately about 400 mg of Holmium oxide. Transfer into a 10 mL volumetric flask containing 5 mL of 1.4 M Perchloric acid solution. Shake the solution well to dissolve Holmium oxide. Make up the volume to the mark with 1.4 M Perchloric acid solution.
• Operate the spectrophotometer according to the SOP on Operation.
• Perform the baseline correction with 1.4 M Perchloric acid solution from 200 to 800 nm.
• Remove the cell from the sample compartment.
• Rinse the sample cell with Holmium perchlorate solution, fill it with the same, clean the smooth surfaces of the cell with tissue paper & place it in the sample compartment then press start.
• Scan it & verify the wavelength using the absorption maxima of the Holmium Perchlorate solution. The permitted tolerance is given in the below table.

Sr. No.	Wavelength	Observed Wavelength	Acceptance Criteria
1	241.15 nm		240.15 to 242.15 nm
2	287.15 nm		286.15 to 288.15 nm
3	361.50 nm		360.50 to 362.50 nm
4	536.30 nm		533.30 to 539.30 nm

Control of Wave Length Using Holmium Oxide Filter:

• Use the Holmium Oxide filter and record the UV-visible absorption spectrum in the range of 200 to 800nm.
• Record the results on the appropriate form.

Control of Absorbance:

• Preparation of 0.005 M Sulphuric acid: Take 100 mL volumetric flask & fill half with water, add 2.8 mL Conc. Sulphuric acid. Dissolve & dilute to the volume with water. Transfer 1 mL of this solution into a 100 mL volumetric flask, dissolve & dilute to the volume with water.
• Preparation of Potassium dichromate (K2Cr2O7) solution: Take about 200 mg of K2Cr2O7 into a weighing bottle & heat at 130°C in an oven for 2 hours. Cool it in a desiccator.
• Transfer accurately weighed about 120 mg in a 100 mL volumetric flask containing 50 mL of 0.005 M Sulfuric acid. Shake the flask well to dissolve Potassium dichromate completely. Make up the volume with 0.005 M Sulfuric acid. Dilute 5mL of the above solution to 100 mL with 0.005M Sulfuric acid.
• Operate the UV Spectrophotometer according to the SOP on operation & use the 0.005M Sulphuric Acid solution as a blank.
• Scan the Potassium dichromate solution between regions 200 – 400 nm & measure the absorbances at 235 nm, 257 nm, 313 nm & 350 nm.
• Calculate the specific absorbance A(1%, 1cm) from the equation.

 

• For the control of absorbance at 430 nm, 57.0 to 63.0 mg of Potassium dichromate →100 mL with 0.005M Sulphuric Acid.

Limit of Stray Light:

• Dry about 3g of Potassium Chloride at 105°C for 1 hour. Cool in a desiccator to room temperature.
• Weigh accurately 1.2 g of the dried potassium chloride & transfer it into a 100 mL volumetric flask. Dissolve with sufficient water & makeup to the mark with the same water. Mix thoroughly.
• Operate the UV Spectrophotometer according to SOP on operation, & use water as a blank.
• Remove the cell from the sample compartment. Rinse the cell in the potassium chloride solution, fill it with the same, clean the smooth surfaces with tissue paper & place it in the sample compartment.
• Measure the absorbance at 200 nm. Acceptance Criteria: The absorbance should be more than 2.

Resolution Power:

• Preparation of 0.02 % v/v of toluene in Hexane solution: Dilute 2 mL of Toluene to 20 mL with Hexane. Further, dilute 0.1 mL of the above solution to 50 mL with Hexane.
• Operate the UV Spectrophotometer according to SOP on operation, & use Hexane as a blank.
• Record the spectrum in the range 260 to 275 nm of 0.02 % v/v toluene in Hexane.
• Calculate the ratio of the absorbance at the maximum at 269 nm to that at the minimum at 266 nm.

Photometric Linearity: 

• Weigh accurately 50mg of Potassium chromate in a 50mL volumetric flask & dissolve in 0.05N Potassium hydroxide solution. Makeup with the same solvent.
• From the above solution take 4mL & make up to 100mL with 0.05N Potassium hydroxide solution.
• Now prepare dilution of 4, 8, 16, 24, 32 μg/mL
• Measure the absorbance at 370nm using 0.05N Potassium hydroxide solution as a blank.
• Acceptance criteria: The plot should be linear & regression coefficient (R2) should be NLT 0.999.

ALSO READ: Operational Qualification of UV Spectrophotometer UV 1800

Baseline Flatness: 

• Operate the UV Spectrophotometer according to the SOP on the operation. Once the UV Probe software is up & running, click on the “Spectrum” button on the toolbar.
• Click on the “Method” button on the toolbar to set up the conditions. A new window will pop up. Set the wavelength range, scan speed & scan interval.

1. Enter wavelength range, start: 800, End: 200
2. Scan Speed: Fast
3. Scan interval: 0.1; Click on OK.

• Click the Baseline. Wait for the baseline correction to be completed.
• Start the scan by clicking on ‘Start’ with air in sample Cells.
• Print the spectrum obtained and compare the baseline flatness to the specification.

Specification	Results
NMT 0.002 Abs

Specification of Cells: 

• Operate the UV Spectrophotometer according to the SOP on the operation. Once the UV Probe software is up & running, click on the “Spectrum” button on the toolbar.
• Click on the “Method” button on the toolbar to set up the conditions. A new window will pop up. Select transmittance mode.
• Set the wavelength at 200 nm. Then press `auto zero’ to get 100 % transmittance with air blank in both the cell holders.
• Then place the cell in the sample compartment filled with DM water using an air blank & read the transmittance.
• Similarly, repeat the operation for 220 nm & 240 nm. Repeat the test for the second cell. The transmittance values should meet the requirement as given in the following table.

Wavelength	Observed % Transmittance	Maximum Tolerance
200 nm		68 to 72 %
220 nm		80 to 84 %
240 nm		85 to 89 %

ATTACHMENTS

Attachments 1: Calibration Report of UV - Visible Spectrophotometer

ABBREVIATIONS

UV – Ultra Violet 

QC – Quality Control 

QA – Quality Assurance 

NMT – Not More Than 

NLT – Not Less Than 

DM – Demineralized 

REVISION HISTORY

Nil

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