UV-Visible Spectrophotometer – Calibration

Standard Operating Procedure (SOP) for Operation and Calibration of UV-Visible Spectrophotometer used for Qualitative and Quantitative analysis of starting material (RM-API & Excipient), water analysis and drug product.

UV-Visible Spectrophotometer

1.0   Objective :

• • To lay down a procedure for operation and calibration of UV-Visible Spectrophotometer.

• • It is used for qualitative & quantitative determination of substances.

2.0   Scope :

• • This SOP is applicable for operation of UV-Visible Spectrophotometer in Quality Control.

3.0   Procedure – UV–Visible Spectrophotometer:

• • Operation of UV–Visible Spectrophotometer:

• • Ensure that equipment is clean.

• • Check the calibration due date.

• • Switch ON the monitor & CPU.

• • Switch ON the equipment.

• • Click twice on the UV probe software icon (UV-Visible Spectrophotometer).

• • Enter the password & click on O.K.

• • Click on the connect button.

• • The screen shows a check list. Instrument check serially all the parameters.

• • When all the points O.K., the passed is marked with green then click on O.K.

• • Now select the Photometric or Spectrum, as required.

• • Photometric Mode of UV–Visible Spectrophotometer:

• • Click on “M” (method) on tool bar.

• • Now Photometric Method Wizard (Wavelength) will appear on screen.

• • Enter the required details and click on NEXT.

• • Now Photometric Method Wizard (Calibration) will appear on screen.

• • Enter the required details and click on NEXT.

• • Enter the details & click on NEXT.

• • Put the analytical details as current date_ product name_ AR. No./ batch No._ test name and click on FINISH.

• • Now click on the CLOSE.

• • Rinse the cuvettes with the solvent used, open the sample cabinet, and place one in sample cabinet & other in reference cabinet. Close the sample cabinet.

• • Click on the auto zero icon & wait till the 0.000 is obtained.

• • Put out one cuvette from the sample cabinet.

• • Rinse the cuvette with solution of which absorbance to be measured and place in the sample cabinet & record the absorbance and save the data in path/file drive D as data path D\UV\current year\current month.

• • After taking the absorbance

• • Click on the Print Icon if you want to print the data.

• • Spectrum Mode of UV–Visible Spectrophotometer:

• • Now Spectrum window will appear on screen.

• • Click on “M” (method) on tool bar.

• • Enter the required details and click on OK.

• • Rinse the cuvettes with the solvent used and place one in sample cabinet & other in reference cabinet.

• • Click on BASELINE ICON, a scanning window will appear on the screen & click OK.

• • Scanning gets started.

• • After completion of scanning put the sample, which is to be scanned then click on START.

• • Window screen open as New data set and ensure file (D/UV/current year/current month/file) and enter
  • the analytical details as current date _product name _AR. No./ batch No._ test name and finish.

• • A scanning graph will represent on the screen.

• • Click on ACTIVE icon to see the single line graph.

• • Then click on OPTION on tool bar and click on PEAK PICK to get the maxima & minima.

• • Click on the Print Icon if you want to print the data

• • After completion of work close the window.

• • Remove the cuvettes and clean with required solvent / Water.

• • Switch off the equipment.

• • Enter the details in Instrument log.

• • Important – UV–Visible Spectrophotometer:

• • Handle cuvettes with care.

• • If non aqueous solvent is used then wash first with Acetone.

• • Hold the cuvette from rough side.

• • For more details refer instruction manual.

• • In case of any deviation inform to service engineer.

• Calibration of UV–Visible Spectrophotometer:

• • Operate the UV–Visible Spectrophotometer as per SOP.

• • Control of Absorbance (UV-Visible Spectrophotometer):

• • Preparation of Potassium Dichromate solution:

• • Use previously dried Potassium Dichromate at 130ºC to constant weight.

• • For the control of absorbance at 235nm, 257 nm, 313 nm, 359 nm, weight accurately a quantity of Potassium Dichromate 57.0 mg – 63.0 mg and dissolve in sufficient 0.005M Sulphuric acid to produce 1000.0 ml.

• • For the control of Absorbance at 430nm dissolve 57.0 to 63.0 mg of Potassium Dichromate in 0.005M Sulphuric Acid and dilute to 100.0 ml.

Related : Calibration of FTIR

• • Wash the unit cells with distilled water and rinse with 0.005M Sulphuric acid.

• • Select the wavelength in the instrument / UV–Visible Spectrophotometer .

• • Fill the unit cells with 0.005M Sulphuric Acid and place them in sample cavity.

• • Adjust zero on screen.

• • Take out the sample cell, rinse with Potassium Dichromate solution and fill the cell with same solution.

• • Place the cell in sample cavity and measure the absorbance at wavelength 235, 257, 313, 350 nm & 430nm and print the data.

• • Now calculate Specific Absorbance (A 1%, 1cm) for Potassium Dichromate solutions using the formula given below.

For wavelength 235, 257, 313 and 350 nm:

                                              Absorbance   x  10000                      

A (1%, 1cm) =—————————————————-

                            Weight of Potassium Dichromate (mg)

For wavelength 430 nm:

                             Absorbance   x  1000                       

A (1%, 1cm) =—————————————————-

                        Weight of Potassium Dichromate (mg)

• • Limits and maximum tolerance are given below

• Control of Wavelength (UV-Visible Spectrophotometer):

  • Holmium Perchlorate Solution:

• • Weigh accurately 1 g. Holmium Oxide and transfer in 25 ml volumetric flask.

• • Add 2.9 ml Perchloric Acid and make up the volume with Milli Q Water.

• • Rinse and fill the cell with Holmium Perchlorate solution and place in the cavity.

• • Scan the solution between 200 to 700 nm at medium scan speed and sampling interval of 0.5.

• • Print the spectrum.

• • Check the maxima at given wavelength given below:

• Limit of stray light (UV-Visible Spectrophotometer) :

• • Weight 1.200g Potassium Chloride and dissolve in sufficient water to produce 0 ml.

• • Rinse and fill the cell with Potassium Chloride solution and place in the cavity.

• • Select the wavelength 200 nm and measure the absorbance using water as blank.

• • The absorbance should not be less than 2.0.

• Resolution Power (UV-Visible Spectrophotometer) :

  • Dilute 1.0 ml toluene to 100.0 ml with n-Hexane.

• • Further dilute 2.0 ml to 100.0 ml with n-hexane.

• • Measure the absorbance at minima 266 nm & maxima 269 nm.

• • The ratio of these two absorbance should not be less than 1.5

• • Cells Pairing:

• • The difference between the internal path lengths of both the cells should not be more than 0.005 cm and measure the path length with the help of vernier caliper.

• • The absorbance of the cells intended to contain the solution to be examined and the reference liquid, when filled with the same solvent should be identical.

• • If this is not the case an appropriate correction must be applied.

• • The tolerance on the path length used is ± 0.005 cm.

• • Solvents:

• • Measure the absorbance of Methanol at 254 nm with reference to water and not exceeding 0.100.

• • Enter the details in Annexure I and enclose the print outs of each test.

• • Prepare the calibration tag and affix it on the equipment.

• • Enclose the old calibration tag with the record and don’t throw it.

• • If the variation is not within the specified tolerance limits:

• • Label the UV-Visible Spectrophotometer with a tag, “OUT OF CALIBRATION”.

• • Inform the Department Head.

• • Do not use equipment until all errors are rectified.

• • After rectification, again calibrate the equipment & record the observation.

Annexure I : Calibration Record of UV-Visible Spectrophotometer

Make : …………………….                                                     Model No.: …………….                               

Frequency: 3 Months ± 3 days                                              Eq. No. : ……………..

Date of Calibration :………………….                                 

• Control of Wavelength:

• Limit of Stray Light :

• Resolution Power :

• Cells Pairing:

• Solvents:

Remarks: The observed values comply / does not comply the acceptance criteria and the equipment can be / cannot be used for routine analysis.

Next calibration due date: ………….

4.0   Reference (S)

• • Chapter No. 2.4.7 of IP 2014

• • Appendix IIB of BP 2015