Calibration of UV/VIS Spectrophotometer
1.0 PURPOSE
The purpose of the standard operating procedure is to calibrate the UV-Visible
Spectrophotometer.
2.0 SCOPE
Standard operating procedure is applicable to the staff responsible for operation and
calibration of the UV-Visible Spectrophotometer.
3.0 RESPONSIBILITY
3.1 Section Incharge
3.2 Technical Manager
3.3 Q.A. Manager
4.0 PROCEDURE
4.1 MATERIALS REQUIRED
4.1.1 Instrument to be used
4.1.2 Potassium dichromate
4.1.3 Sulphuric acid
4.1.4 Potassium chloride
4.1.5 Toluene
4.1.6 Hexane
4.2 CONTROL OF ABSORBANCE
Prepare a solution of potassium dichromate solution, which has been previously dried to constant weight at 130oC using 57.0 to 63.0mg in sufficient 0.005M Sulphuric acid to make up to volume of 1000ml.
Determine the absorbance of the prepared potassium dichromate at different wavelengths of 235nm, 257nm, 313nm and 350nm.
Based on the absorbance obtained, at the particular wavelengths, determine the value of A (1%, 1cm) at each wavelength.
The IP-1996 gives the following tolerances:
| S. No. | Wavelength (nm) | A (1%, 1cm) | Maximum Tolerance |
| 1. | 235 | 124.5 | 122.9 to 126.2 |
| 2. | 257 | 144.5 | 142.8 to 146.2 |
| 3. | 313 | 48.6 | 47.0 to 50.3 |
| 4. | 350 | 107.3 | 105.6 to 109.0 |
4.3 LIMIT OF STRAY LIGHT:
Stray light is detected at 200 nm with 1.2 % w/v solution of potassium chloride at a path
length of 1 cm.
The absorbance of 1.2 % w/v solution of potassium chloride should be greater than 2.0
when compared with water as reference liquid.
4.4 RESOLUTION POWER:
Prepare a solution of 0.02 % v/v of Toluene in hexane.
Take the absorbance at 269 nm and 266 nm.
The ratio of absorbance at the maximum (269 nm) to that at the minimum (266 nm) is
not less than 1.5.
0 Comments: