# 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.