Calibration of UV visible Spectrophotometer | Calibration of UV Spectrophotometer |
- UV Spectrophotometer SOP covers below points:
- Precautions
- Calibration parameters of uv visible spectrophotometer
- Control of wavelengths
- Control of absorbance
- Limit of stray light
- Resolution power
- Solvents
- Cells
- UV visible spectrophotometer Calibration format
1.0 OBJECTIVE :
- To provide a procedure for Calibration of UV Spectrophotometer.
2.0 SCOPE :
- This procedure is applicable to UV Spectrophotometer.
3.0 RESPONSIBILITY :
- Quality Control Chemist and above
4.0 ACCOUNTABILITY:
- Quality Control Head
5.0 PROCEDURE :
Ultraviolet-visible spectroscopy refers to absorption spectroscopy in the ultraviolet-visible spectral region. This means it uses light in the visible and adjacent (near-UV and near-infrared (NIR) ranges. Ultraviolet-visible spectrophotometer is used in the quantitative determination of concentrations of the absorber in the solutions of transition metal ions and highly conjugated organic compounds.
5.1 PRECAUTIONS
- Place the instrument as far as possible away from any strong magnetic or electric field or any electrical apparatus generating a high frequency.
- Avoid placing it where severe vibrations exist or when the sun beats.
- Place the instrument free from dust and corrosive gases.
- To extend the life of the source lamp, have them powered only when required.
- Cells should be cleaned and handled with great care. Wipe the cell properly with tissue or dry soft cloth.
- Never use alkali, abrasive / etching material or hot concentrated acids.
5.2 CALIBRATION
- CONTROL OF WAVELENGTHS
- Solution Preparation
- 1.4 M Perchloric Acid Solution: Take 11.5 ml of Perchloric acid (about 60% w/w strength) in to a clean 100 ml volumetric flask. Dilute to the volume with distilled water (Note: Dilute 8.5 ml to 100 ml with distilled water in case of 70% w/w strength of Perchloric acid).
- Holmium Perchlorate Solution: Weigh accurately about 400 mg 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 and make up the volume to the mark with 1.4 M Perchloric acid solution.
- Procedure
- Take a pair of cuvette having a path length of 1 cm. Perform the base line correction with 1.4 M Perchloric acid solution from 200 to 600 nm.
- Remove the cell from sample compartment.
- Rinse the sample cell with Holmium perchlorate solution, fill the cell with the same, clean the smooth surfaces of the cell with tissue paper and place it in the sample compartment and record the spectra of solution.
- Acceptance Criteria:
- The permitted tolerance is ± 1 nm for the range 200 nm to 400 nm and ± 3 nm for the range of 400 nm to 600 nm.
- Solution Preparation
| Wavelength | Maximum Tolerance |
| 241.15 nm | 240.15 to 242 .15 nm |
| 287.15 nm | 286.15 to 288.15 nm |
| 361.50 nm | 360.50 to 362 .50 nm |
| 536. 30 nm | 533.30 to 539.30 nm |
- CONTROL OF ABSORBANCE
- Solution Preparation
- 0.005 M Sulphuric Acid: Dilute 0.41 ml of Conc. Sulphuric acid to 1500 ml with water.
- Weigh and transfer about 0.5 gm of Potassium Dichromate (Primary standard) in to a weighing bottle and dry at 130oC in a laboratory oven and cool it in a desiccator.
- Solution A: Weigh accurately quantity NLT 57.0 mg and NMT 63.0 mg and dissolve in 1000 ml volumetric flask containing 300 ml of 0.005 M Sulphuric acid. Shake the flask well to dissolve Potassium dichromate completely. Make up the volume with 0.005 M Sulphuric acid.
- Solution B: Weigh accurately quantity NLT 57.0 mg and NMT 63.0 mg and dissolve in 100 ml volumetric flask containing 50 ml of 0.005 M Sulphuric acid. Shake the flask well to dissolve Potassium dichromate completely. Make up the volume with 0.005 M Sulphuric acid.
- Procedure
- Take a pair of cuvette having a path length of 1 cm.
- Perform base line correction with 0.005 M Sulphuric acid from 200 to 600 nm. Remove the cell from sample compartment. Rinse the sample cell with solution A, fill the cell with the same, clean the smooth surfaces with tissue paper. Place the cell in sample compartment, record the spectra at 200 to 600 nm and measure the absorbance at 235 nm, 257 nm, 313 nm and 350 nm.
- Rinse the sample cell with solution B, fill the cell with the same, clean the smooth surfaces with tissue paper. Place the cell in sample compartment, measure the absorbances at 430 nm.
- Calculation
- Calculate the specific absorbance using the following equation.
- Specific absorbance at x nm = Absorbance at x nm X final volume X 10 / weight of sample (mg)
- Acceptance Criteria
- The specific absorbance against each wavelength should be as follows:
- Solution Preparation
| Wavelength ( x nm) | Specific absorbance (1%, 1 cm) | Maximum tolerance |
| 235 | 124.5 | 122.9 to 126.2 |
| 257 | 144.5 | 142.8 to 146.2 |
| 313 | 48.6 | 47.0 to 50.3 |
| 350 | 107.3 | 105.6 to 109.0 |
| 430 | 15.9 | 15.7 to 16.1 |
- LIMIT OF STRAY LIGHT
- Solution Preparation
- 1.2% w/v Potassium Chloride Solution:
- Weigh accurately about 1.2 gm of Potassium chloride and transfer into a 100 ml volumetric flask containing 75 ml distilled water. Shake the flask well to dissolve completely and make up to the volume with distilled water, put stopper and shake again.
- Procedure
- Take a pair of cuvette having a path length of 1 cm.
- Perform the auto zero with distilled water. Remove the cell from sample compartment. Rinse the cell in the potassium chloride solution, fill the cell with the same, clean the smooth surfaces with tissue paper and place it in sample compartment.
- Measure the absorbance at 200 nm.
- Acceptance Criteria:
- The absorbance is not less than 2.
- Solution Preparation
- RESOLUTION POWER
- Solution Preparation
- 0.02 % v/v of toluene in Hexane: Dilute 2 ml of Toluene to 200 ml with Hexane. Further dilute 2 ml of resulting solution to 100 ml with Hexane.
- Procedure
- Record the spectra in the range of 260 to 275 nm using Hexane in the reference cell.
- Calculation
- Ratio of the absorbance = Absorbance at 269 nm / Absorbance at 266 nm
- Acceptance Criteria :
- Ratio of absorbance is not less than 1.5.
- Solution Preparation
- LINE FLATNESS
- Procedure
- Perform the test with air blank in the reference and sample compartment.
- Scan in the wavelength range 220 to 700 nm.
- Obtain the spectrum in % transmittance mode.
- Acceptance Criteria
- The line obtained should not deviate from a horizontal line at any point by more than 98.0% and less than 102.0% transmittance.
- Procedure
- SOLVENTS
- Procedure
- Take the absorbance of following solvents at about 254 nm with reference to distilled water.
- Ethanol (95%)
- Ethanol
- Methanol
- Cyclohexane
- Take the absorbance of following solvents at about 254 nm with reference to distilled water.
- Acceptance Criteria: Absorbance should not more than 0.10
- Procedure
- CELLS
- Procedure
- Perform the auto zero with air blank in both the cell holders.
- Set the wavelength at 200 nm.
- Then place the cell in the sample compartment filled with distilled water using air blank in reference compartment and measure the absorbance.
- Similarly repeat the procedure for 220 nm and 240 nm.
- Repeat the test for second cell.
- Absorbance of both cells should be same.
- Acceptance Criteria
- The absorbance difference between two cells should not more than ± 0.01.
- Procedure
6.0 ABBREVIATIONS:
| Abbreviation | Expanded form |
| UV | Ultra Violet |
| nm | Nanometer |
| Mg | Miligram |
| Conc. | Concentrated |
| gm | Gram |
| cm | Centimeter |
| NMT | Not more than |
| NLT | Not less than |
| AR grade | Analytical Reagent Grade |
| % | Percentage |
| w/w | Weight by Weight |
| w/v | Weight by Volume |
7.0 ANNEXURES:
| Annex. No. | Title |
| 01 | Calibration of Ultraviolet Visible Spectrophotometer |
8.0 SOP REFERENCES
- Indian Pharmacopoeia
END OF THE SOP
ANNEXURES :
Annex. No. 01 Calibration of Ultraviolet Visible Spectrophotometer
