BACKGROUND
Ultraviolet Spectrophotometer
Ultraviolet Spectrophotometer is used to find out various technical information related to absorbance, wavelength of chemicals, hence its calibration is necessary to acquire precise and accurate results.1,2
REQUIREMENTS
- Ultraviolet Spectrophotometer
- Holmium Oxide
- Perchloric acid
- Potassium dichromate
- Hot air oven
- Weighing Balance
- Volumetric flask – 100 and 1000 ml
- 0.005 M Sulphuric acid
- Potassium chloride
- Water
- Toluene
- Hexane
PROCEDURE
Control of wavelength
Add 1.0 g of Holmium Oxide in 1.4 M perchloric acid, make up to 25 ml with perchloric acid ensuring proper mixing. Perform the baseline correction by taking only 1.4 M percholric acid liquid in the reference and sample cuvette and perform auto zero. Take holmium oxide solution in sample cuvette and start scan (range 200 to 800) to get absorption spectrum. Control of Wavelength is ascertained with the following acceptance criteria. Deviations should not be greater than ± 3 nm in the visible range and ± 1 nm in the ultra-violet.
Acceptance criteria
| Sl. No. | Maxima at wavelength (nm) | Wavelength nm | Deviation allowed |
| Peak 1 | 241.15 | 240.15 to 242.15 | ± 1 nm |
| Peak 2 | 287.15 | 286.15 to 288.15 | ± 1 nm |
| Peak 3 | 361.5 | 360.5 to 362.5 | ± 1 nm |
| Peak 4 | 536.3 | 533.3 to 539.3 | ± 3 nm |
Control of absorbance
Take about 100 mg of potassium dichromate and dry at 130°C in hot air oven. Weight accurately 60 mg of potassium dichromate and pour in a 1000 ml volumetric flask / suitable glass container containing about 400 ml of 0.005 M sulphuric acid, stir well to mix and make up the volume up to 1000 ml by 0.005 M sulphuric acid. This solution (A) is for ultraviolet region to be measured at 235, 257, 313 and 350 nm.
Again, Weight accurately 60 mg of potassium dichromate and pour in a 100 ml volumetric flask / suitable glass container containing about 40 ml of 0.005 M sulphuric acid, stir well to mix and make up the volume up to 100 ml by 0.005 M sulphuric acid. This solution (B) is for visible region to be measured at 430 nm.
Perform the baseline correction by taking only 0.005 M sulphuric acid liquid in the reference and sample cuvette and perform auto zero.
Take absorbance readings with solution A, clean cuvette and repeat with solution B.
Acceptance criteria
| Sl No. | Wavelength (nm) | Specific AbsorbanceA 1 %, 1cm | Maximum Tolerance |
| 1 | 235 (UV range) | 124.5 | 122.9 – 126.2 |
| 2 | 257 (UV range) | 144.5 | 142.8 – 146.2 |
| 3 | 313(UV range) | 48.6 | 47.0 – 50.3 |
| 4 | 350 (UV range) | 107.3 | 105.6 – 109.0 |
| 5 | 430 (Visible range, use solution B) | 15.9 | 15.7 – 16.1 |
Limit of stray light
Take 1.2 g of potassium chloride (dried) in 100 ml volumetric flask containing about 50 ml of distilled water, mix well and makeup the volume up to 100 ml with water. Perform the baseline correction by taking only water in the reference and sample cuvette and perform auto zero.
Now measure the absorbance of potassium chloride solution at 198 nm.
Acceptance criteria
Absorbance should be more than 2.0
Resolution power
Take 0.2 ml of toluene in 1000 ml volumetric flask and make up the volume by hexane. Perform the baseline correction by taking only hexane in the reference and sample cuvette and perform auto zero.
Now measure the absorbance of solution (toluene in hexane).
Record the spectrum of the above solution from 230 to 330 nm, and note the absorbance at 266 and 269 nm.
Acceptance criteria
Ratio of the absorbance at the 269 nm maximum to that at the 266 nm minimum should be greater than 1.5.
CONCLUSION
The calibration of Ultraviolet Spectrophotometer is helpful to get correct readings and should be performed in routine manner.
REFERENCES
- http://www.pharmaguideline.com/2011/02/sop-for-calibration-of-uv-vis.html
- http://qualitysquare.blogspot.in/2012/10/of-uv-visible-spectrophotometer.html
Also read:
- Calibration of pH meter
- Assay of Azithromycin by HPLC in tablet dosage form
- Assay of Cefadroxil by HPLC in tablet dosage form
- Preparation and standardization of sulfuric acid
- Determination of the concentration of potassium ion using Flame Photometry
- Preparation and standardization of ceric ammonium sulphate
- Acute toxicity testing for topical preparations
- Calculation of Body Surface Area (Using a Nomogram)
- Estimation of white blood cell count (TLC/ Total Leucocyte Count)
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