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.UV-Visible Spectrophotometer
    • 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
Wavelength (nm) Specific Absorbance Maximum Tolerance
235 124.5 122.9 – 126.2
257 144.5 142.8 – 146.2
313 48.6 47.0 – 50.3
350 107.3 105.6 – 109.0
430 15.9 15.7 – 16.1
  • 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:
Wavelength (nm) Maximum Tolerance (nm)
241.15 240.15 – 242.15
     287.15 286.15 – 288.15
    361.5 360.5 – 362.5
    536.3 533.3 – 539.3
  • 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:
    • 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 Absorbance:
Weight of K2Cr2O7 :…………….. mg (W1) dissolve in 1000.0 ml of 0.005 M H2SO4

Weight of K2Cr2O7 :……………. mg (W2) dissolve in 100.0 ml of 0.005 M H2SO4    ( for 430 nm)

Wave length Observed value of Tolerance
Absorbance (Aº) Specific Absorbance (A 1%, 1cm)
Calculation Result
235 nm   ……………..(A0)x10000

…………. (W1)

  122.9 to 126.2
257 nm   ……………..(A0)x10000

…………. (W1)

  142.8 to 146.2
313 nm   ……………..(A0)x10000

…………. (W1)

  47.0 to 50.3
350 nm   ……………..(A0)x10000

…………. (W1)

  105.6 to 109.0
430 nm   ……………..(A0)x1000

…………. (W2)

  15.7 to 16.1
  • Control of Wavelength:

Weigh accurately 1 g. (……………….) Holmium Oxide and transfer in 25 ml volumetric flask. Add 2.9 ml Perchloric Acid (…………….ml) and make up the volume with Milli Q Water (…………ml).
Standard Wavelength Observed Reading Tolerance Limit
241.15   240.15 – 242.15
287.15   286.15 – 288.15
361.50   360.50 – 362.50
536.30   533.30 – 539.30
  • Limit of Stray Light :

Weight 1.200g Potassium Chloride (…………… g) and dissolve in sufficient distilled water to produce   100.0 ml (………………ml).
Wavelength Observed absorbance (Aº) Limit (Aº)
200 nm Not Less than 2.0
  • Resolution Power :

Dilute 1.0 ml (           ml) toluene to 100.0 ml (            ml) with n-Hexane. Further dilute 2.0 ml                         (          ml) to 100.0 ml (                       ml) with n-hexane.
Wavelength Observed absorbance Aº Observed Ratio Limit
269 nm NLT 1.5
  • Cells Pairing: 
 Sr. No.  Path length of Cell 1  Path length of cell 2  Difference

Limit ± 0.005 cm

Remarks
  • Solvents: 
Wavelength Cell No. Absorbance of Methanol with Reference to Air Limit

NMT

0.2

Absorbance of Methanol with Reference to Water Limit

NMT

0.1

Remarks
254 nm Cell 1          
254 nm Cell 2          

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

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