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 :
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- To lay down a procedure for operation and calibration of UV-Visible Spectrophotometer.
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- It is used for qualitative & quantitative determination of substances.
2.0 Scope :
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- This SOP is applicable for operation of UV-Visible Spectrophotometer in Quality Control.
3.0 Procedure – UV–Visible Spectrophotometer:
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Operation of UV–Visible Spectrophotometer:
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- Ensure that equipment is clean.
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- Check the calibration due date.
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- Switch ON the monitor & CPU.
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- Switch ON the equipment.
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- Click twice on the UV probe software icon (UV-Visible Spectrophotometer).
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- Enter the password & click on O.K.
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- Click on the connect button.
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- The screen shows a check list. Instrument check serially all the parameters.
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- When all the points O.K., the passed is marked with green then click on O.K.
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- Now select the Photometric or Spectrum, as required.
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Photometric Mode of UV–Visible Spectrophotometer:
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- Click on “M” (method) on tool bar.
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- Now Photometric Method Wizard (Wavelength) will appear on screen.
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- Enter the required details and click on NEXT.
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- Now Photometric Method Wizard (Calibration) will appear on screen.
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- Enter the required details and click on NEXT.
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- Enter the details & click on NEXT.
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- Put the analytical details as current date_ product name_ AR. No./ batch No._ test name and click on FINISH.
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Now click on the CLOSE.
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- 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.
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- Click on the auto zero icon & wait till the 0.000 is obtained.
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- Put out one cuvette from the sample cabinet.
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- 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.
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- After taking the absorbance
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- Click on the Print Icon if you want to print the data.
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Spectrum Mode of UV–Visible Spectrophotometer:
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- Now Spectrum window will appear on screen.
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- Click on “M” (method) on tool bar.
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- Enter the required details and click on OK.
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- Rinse the cuvettes with the solvent used and place one in sample cabinet & other in reference cabinet.
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- Click on BASELINE ICON, a scanning window will appear on the screen & click OK.
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- Scanning gets started.
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- After completion of scanning put the sample, which is to be scanned then click on START.
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- 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.
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- A scanning graph will represent on the screen.
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- Click on ACTIVE icon to see the single line graph.
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- Then click on OPTION on tool bar and click on PEAK PICK to get the maxima & minima.
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- Click on the Print Icon if you want to print the data
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- After completion of work close the window.
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- Remove the cuvettes and clean with required solvent / Water.
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- Switch off the equipment.
- Switch off the equipment.
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- Enter the details in Instrument log.
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Important – UV–Visible Spectrophotometer:
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- Handle cuvettes with care.
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- If non aqueous solvent is used then wash first with Acetone.
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- Hold the cuvette from rough side.
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- For more details refer instruction manual.
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- In case of any deviation inform to service engineer.
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Calibration of UV–Visible Spectrophotometer:
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- Operate the UV–Visible Spectrophotometer as per SOP.
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Control of Absorbance (UV-Visible Spectrophotometer):
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- Preparation of Potassium Dichromate solution:
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- Use previously dried Potassium Dichromate at 130ºC to constant weight.
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- 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.
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- 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
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- Wash the unit cells with distilled water and rinse with 0.005M Sulphuric acid.
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- Select the wavelength in the instrument / UV–Visible Spectrophotometer .
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- Fill the unit cells with 0.005M Sulphuric Acid and place them in sample cavity.
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- Adjust zero on screen.
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- Take out the sample cell, rinse with Potassium Dichromate solution and fill the cell with same solution.
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- Place the cell in sample cavity and measure the absorbance at wavelength 235, 257, 313, 350 nm & 430nm and print the data.
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- 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)
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- 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 |
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Control of Wavelength (UV-Visible Spectrophotometer):
- Holmium Perchlorate Solution:
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- Weigh accurately 1 g. Holmium Oxide and transfer in 25 ml volumetric flask.
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- Add 2.9 ml Perchloric Acid and make up the volume with Milli Q Water.
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- Rinse and fill the cell with Holmium Perchlorate solution and place in the cavity.
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- Scan the solution between 200 to 700 nm at medium scan speed and sampling interval of 0.5.
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- Print the spectrum.
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- 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 |
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Limit of stray light (UV-Visible Spectrophotometer) :
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- Weight 1.200g Potassium Chloride and dissolve in sufficient water to produce 0 ml.
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- Rinse and fill the cell with Potassium Chloride solution and place in the cavity.
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- Select the wavelength 200 nm and measure the absorbance using water as blank.
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- The absorbance should not be less than 2.0.
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Resolution Power (UV-Visible Spectrophotometer) :
- Dilute 1.0 ml toluene to 100.0 ml with n-Hexane.
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- Further dilute 2.0 ml to 100.0 ml with n-hexane.
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- Measure the absorbance at minima 266 nm & maxima 269 nm.
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- The ratio of these two absorbance should not be less than 1.5
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Cells Pairing:
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- 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.
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- 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.
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- If this is not the case an appropriate correction must be applied.
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- The tolerance on the path length used is ± 0.005 cm.
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Solvents:
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- Measure the absorbance of Methanol at 254 nm with reference to water and not exceeding 0.100.
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- Enter the details in Annexure I and enclose the print outs of each test.
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- Prepare the calibration tag and affix it on the equipment.
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- Enclose the old calibration tag with the record and don’t throw it.
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- If the variation is not within the specified tolerance limits:
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- Label the UV-Visible Spectrophotometer with a tag, “OUT OF CALIBRATION”.
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- Inform the Department Head.
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- Do not use equipment until all errors are rectified.
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- 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 :………………….
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| 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) |
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| 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 | ||
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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 | |
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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 | |
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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)
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- Chapter No. 2.4.7 of IP 2014
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- Appendix IIB of BP 2015
